Lilrb2 and notch-mediated expansion of hematopoietic precursor cells

ABSTRACT

The current disclosure describes methods of expanding precursor cells for hematopoietic transplantation in subjects. The methods culture precursor cells in media containing an immobilized high molecular weight LILRB2 agonist or an LILRB2 agonist in combination with a Notch agonist. The expanded cells can be used to treat a variety of hematopoietic disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/313,043, filed on Nov. 21, 2016, which is a national phase based onInternational Application No. PCT/US2015/031959, filed on May 21, 2015,which claims the benefit of U.S. Provisional Application No. 62/002,101,filed on May 22, 2014, and U.S. Provisional Application No. 62/005,746,filed on May 30, 2014, each of which is incorporated herein by referencein its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 24B4779_ST25.txt. The text file is 79.0 KB, wascreated on Jun. 24, 2019, and is being submitted electronically viaEFS-Web.

FIELD OF THE DISCLOSURE

The current disclosure describes methods of expanding precursor cellsfor hematopoietic transplantation in subjects. The methods cultureprecursor cells in media containing an immobilized high molecular weightLILRB2 agonist or an LILRB2 agonist in combination with a Notch agonist.The expanded cells can be used to treat a variety of hematopoieticdisorders.

BACKGROUND OF THE DISCLOSURE

Hematopoietic stem cells (HSC) are pluripotent and ultimately gives riseto all types of terminally differentiated blood cells. HSC canself-renew or differentiate into more committed hematopoietic progenitorcells (HPC), which progenitor cells are irreversibly determined to beancestors of only a few types of blood cell. For instance, HSC candifferentiate into (i) myeloid progenitor cells, which myeloidprogenitor cells ultimately give rise to monocytes and macrophages,neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells, or (ii) lymphoid progenitorcells, which lymphoid progenitor cells ultimately give rise to T-cells,B-cells, and lymphocyte-like cells called natural killer cells(NK-cells). Once the HSC differentiate into a myeloid progenitor cell,its progeny cannot give rise to cells of the lymphoid lineage, and,lymphoid cells cannot give rise to cells of the myeloid lineage. For ageneral discussion of hematopoiesis and HSC differentiation, see Chapter17, Differentiated Cells and the Maintenance of Tissues, Alberts et al.,1989, Molecular Biology of the Cell, 2nd Ed., Garland Publishing, NewYork, N.Y.; Chapter 2 of Regenerative Medicine, Department of Health andHuman Services, Aug. 5, 2006, and Chapter 5 of Hematopoietic Stem Cells,2009, Stem Cell Information, Department of Health and Human Services.Precursor cells can include HSC, HPC and/or mixtures of HSC and HPC.

Precursor cell transplantation represents an important therapy due tothese cell's capacity to restore blood and immune cells in transplantrecipients. For example, transplantation of precursor cells can be usedto treat subjects with inherited immuno-deficient or autoimmune diseasesand diverse hematopoietic disorders. Precursor cell transplantation canalso be used to treat chemotherapy and radiation-treatment patientsbecause prolonged neutropenia and pancytopenia is common following thesetreatment regimens. As one example, and of particular concern,chemotherapeutic treatments for AML result in prolonged periods ofprofound neutropenia with infectious complications still common even inthe setting of modern antimicrobial therapies with mortality rates ashigh as 20% in adolescent and young adults. Human bone marrowtransplantation methods are also currently used as therapies forleukemia, lymphoma, and other life-threatening diseases.

In transplantation, it has been observed that patients receiving greaternumbers of expanded precursor cells have more rapid recovery of theirneutrophils following transplantation. Accordingly, high doses ofprecursor cells are needed to achieve rapid and sustained engraftmentthat is critical for a patient's survival and recovery. These findingssuggest a critical need for generating greater numbers of precursorcells that reliably enhance neutrophil recovery.

Although progress toward efficient ex vivo expansion of precursor cellshas been made, significant improvements in the efficacy andreproducibility of this technology are needed before it can be widelyused. Accordingly, new approaches are needed.

SUMMARY OF THE DISCLOSURE

Described herein are methods of expanding precursor cells ex vivo,comprising culturing said precursor cells with a LILRB2 agonistimmobilized on a first solid phase, wherein the immobilized LILRB2agonist is (i) an antibody to the LILRB2 receptor, or (ii) anantigen-binding fragment of said antibody; and wherein the precursorcells are hematopoietic stem cells or hematopoietic progenitor cells. Incertain embodiments, the methods described herein comprise culturingsaid precursor cells with an immobilized LILRB2 agonist in combinationwith a Notch agonist. In specific embodiments, the precursor cells arehuman cells. In more specific embodiments, the precursor cells areobtained from bone marrow, umbilical cord blood, placental blood, orWharton's jelly. In more specific embodiments, the precursor cells areobtained from fetal or neonatal blood.

In certain embodiments, the antibody to the LILRB2 receptor is amonoclonal antibody. In certain embodiments, the antibody to the LILRB2receptor is a polyclonal antibody. In certain embodiments, the LILRB2agonist is an Fv, Fab, Fab′, F(ab′)₂, Fc, or single chain Fv fragment(scFv). In certain embodiments, the antibody to LILRB2 is a human,humanized, synthetic, or chimeric antibody. In specific embodiments, theLILRB2 agonist binds to the Ig1 domain of LILRB2. In specificembodiments, the LILRB2 agonist binds to the Ig4 domain of LILRB2. Inspecific embodiments, the LILRB2 agonist binds to the Ig1 and Ig4domains of LILRB2.

In certain embodiments, the first solid phase is the surface of a tissueculture dish. In certain embodiments, the Notch agonist is immobilizedon the first solid phase. In certain embodiments, the Notch agonist isimmobilized on a second solid phase that is not the first solid phase.In certain embodiments, the Notch agonist is immobilized on the firstsolid phase, and the first solid phase is the surface of a tissueculture dish. In certain embodiments, the Notch agonist is immobilizedon a second solid phase that is not the first solid phase, wherein thefirst solid phase is the surface of a tissue culture dish or flask, andthe second solid phase is a bead. In certain embodiments, the Notchagonist is immobilized on a second solid phase that is not the firstsolid phase, wherein the first solid phase is a bead, and the secondsolid phase is the surface of a tissue culture dish or flask.

In certain embodiments, the Notch agonist is an extracellular,Notch-interacting domain of a Delta protein. In specific embodiments,the Notch agonist is human Delta-1. In specific embodiments, the Notchagonist is Delta^(ext-IgG). In more specific embodiments, the Notchagonist is in dimeric form. In specific embodiments, the Notch agonistis an antibody that specifically binds to a Notch motif. In morespecific embodiments, the Notch agonist is an antibody that specificallybinds to Notch-1. In specific embodiments, the Notch agonist is anantibody that specifically binds to Notch-2.

In certain embodiments, the culturing step is performed in the presenceof a culture medium comprising stem cell factor (SCF), thrombopoietin(TPO), and Flt3-ligand. In specific embodiments, the culturing step isperformed in the presence of a culture medium comprising 10-100 ng/mLSCF, 5-100 ng/mL TPO and 10/100 ng/mL Flt3-ligand. In certainembodiments, the culture medium comprises 50 ng/mL SCF. In certainembodiments, the culture medium comprises 10 ng/mL TPO. In certainembodiments, the culture medium comprises 50 ng/mL Flt3-ligand. Incertain embodiments, the culture medium comprises 50 ng/mL SCF, 10 ng/mLTPO and 50 ng/mL Flt3-ligand.

In certain embodiments, the culturing step is performed in the presenceof a culture medium comprising SCF, Flt3-ligand, interleukin-6 (IL-6),TPO, fibroblast growth factor-1 (FGF1), and interleukin-3 (IL-3). Inspecific embodiments, the culture medium comprises SCF, Flt3-ligand,IL-6, TPO, FGF1, IL-3, and heparin. In certain embodiments, the culturemedium comprises 1-100 ng/mL SCF, 1-100 ng/mL Flt3-ligand, 1-100 ng/mLIL-6, 1-100 ng/mL TPO, 1-100 ng/mL FGF1, and 1-100 ng/mL IL-3. Inspecific embodiments, the culture medium further comprises 1-100 μg/mLheparin. In certain embodiments, the culture medium comprises 50 ng/mLSCF, 50 ng/mL Flt3-ligand, 50 ng/mL IL-6, 50 ng/mL TPO, 20 ng/mL FGF1,and 10 ng/mL IL-3. In specific embodiments, the culture medium furthercomprises 10 μg/mL heparin.

In certain embodiments, the culturing step is performed in the presenceof a culture medium comprising retronectin. In specific embodiments, theculture medium comprises 1-100 μg/mL retronectin. In more specificembodiments, the culture medium comprises 5 μg/mL retronectin.

The current disclosure provides improved methods to generate precursorcells through ex vivo expansion. The methods generate more precursorcells more quickly than previously-available methods and the generatedprecursor cells show enhanced early marrow repopulation inimmuno-deficient subjects and improved long term repopulation followingtransplant. The methods disclosed herein are based on culturingprecursor cells with an immobilized LILRB2 agonist or an LILRB2 agonistin combination with a Notch agonist.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1H. High molecular weight Angptl2 activates LILRB2 signaling.(FIG. 1A) Schematic of the chimeric LILRB2 receptor reporter system.(FIG. 1B) Representative flow cytometric profiles and summary showingthat the Angptl2 conditioned medium stimulates GFP induction in theLILRB2 chimeric reporter system. The condition media of emptyvector-transfected HEK-293T cells was used as control. (FIG. 1C) Left,secreted Angplt2 and HLA-G-ECD in condition medium detected by anti-FLAGantibody in Western blotting. Right, representative flow cytometricplots showing that Angptl2 binds to LILRB2 expressed on HEK-293T cellsbetter than the same amount of HLA-G-ECD. (FIG. 1D) The full-length(FL), coil-coil domain (CC), and fibrinogen domain (FBN) obtained fromconditioned medium showed distinctive migration in reducing andnon-reducing SDS-PAGE as determined by immunoblotting with anti-M2 Flagantibody. Protein extracted from equivalent amounts of condition mediaof empty vector-transfected HEK293T cells was used as control. (FIG. 1E)GST-human Angptl2 purified from bacterial expression system byGlutathione Sepharose was immediately fractionated through gelfiltration FPLC. The molecular weight was determined by the peaks ofApoferritin (443 KD), Amylase (200 KD), Alcohol dehydrogenase (150 KD),Albumin (66 KD), Carbonic anhydrase (29 KD), and Cytochrome c (12.4 KD),respectively. (FIG. 1F) Equivalent amounts of indicated fractionatedsamples in FPLC were loaded on 10% native gel. Aggregated, monomeric,and cleaved GST-Angptl2 were visualized by silver staining. (FIG. 1G)Indicated FPLC fractionated samples were examined by Western blottingusing antiM2 Flag antibody. The FLAG in cleaved GST-Angptl2 fragments(fraction 8; FIG. 1G) could not be detected by Western blotting. (FIG.1H) Chimeric LILRB2 receptor reporter cells were treated with coated orsoluble fraction 5 proteins for 48 hrs. In coated wells, 5 μg/mlGST-Angptl2 from fraction-5 was pre-coated onto wells of a 96-well platefor 3 hrs at 37° C. Equivalent amount of FPLC buffer was used ascontrol. n.s. indicates not significant; ****, p<0.0001.

FIGS. 2A-2B. Schematic of Angptl2, Angptl5, and HLA-G extracellulardomain (ECD) expression constructs. (FIG. 2A) Constructs for secretableAngptl2, Angptl5, and HLA-G-ECD. Angptls without signal peptide (SP) orHLA-G ECD was fused to an optimized signal peptide(MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 1)) at the N terminus and a FLAG tagat the C terminus. (FIG. 2B) FACS plots showing the binding of Angptl5to LILRB2 expressing 293T cells.

FIG. 3. Activation of the LILRB2 chimeric receptor reporter by Angptl2is better than that by immobilized HLA-G. Chimeric LILRB2 receptorreporter cells were treated with coated 5 μg/ml GST-Angptl2 or 130 μg/mlhuman HLA-G for 24 hrs. Null receptor cells, which do not containchimeric receptor, and PBS were used as controls.

FIG. 4. Expression of mAngptl2 in mouse tissues and organs. Whole celllysates were extracted from mouse spleen, fat, muscle, heart, brain andkidney. Plasma and serum were extracted from peripheral blood viacentrifugation with or without anti-clotting reagent, respectively.Fifty μg of each sample was loaded for western blot using rat anti-mouseAngptl2 antibody. The samples loaded were visualized by CoomassieBrilliant Blue staining (CBB). R, reduced; N, non-reduced; M, molecularweight marker. Arrows indicate the multimerized mouse Angptl2.

FIGS. 5A-5D. Immobilized anti-LILRB2 antibodies activated the chimericLILRB2 reporter. (FIG. 5A) Representative flow cytometric profilesshowing that the GFP induction by immobilized 5 μg/ml Angptl2 wasabolished by 5 μg/ml anti-LILRB2 antibody. Chimeric LILRB2 receptorreporter cells were treated with indicated coated Angptl2 with orwithout soluble anti-LILRB2 pAb or mAb for 48 hrs. PBS was used ascontrol. (FIG. 5B) Representative flow cytometric profiles showing thatGFP was induced by immobilized anti-LILRB2 antibodies. Chimeric LILRB2receptor reporter cells were treated with indicated coated (25 μg/ml in50 μl PBS) or soluble (5 μg/ml in 250 μl cell culture media) antibodiesfor 48 hrs. The reporter cells not containing chimeric LILRB2 receptorwere used as negative control. (FIG. 5C) Representative flow cytometricprofiles showing that GFP expression was induced by cross-linkedanti-LILRB2 antibodies. Chimeric LILRB2 receptor reporter cells weretreated with 10 μg/ml soluble anti-LILRB2 polyclone antibody (pAb) orequivalent crosslinked pAb for 48 hrs. Streptavidin alone was used as anegative control. (FIG. 5D) Representative confocal images of LILRB2chimeric receptor reporter cells with or without coated anti-LILRB2 mAbshowing that the distribution of LILRB2 protein on cell plasma membrane.Ten confocal scans from top to bottom of a cell were indicated fromLayer-1 (L1) to Layer-10 (L10). Confocal images of the phase contrast,Cy3 (indicating LILRB2 expression), and GFP (indicating signalingactivation) panels were merged.

FIGS. 6A-6I. Ig domains 1 and 4 in LILRB2 are critical for Angptl2binding and signal activation. (FIG. 6A) Representative flow cytometryplots showing Angptl2 binding to full-length, individual Ig domain,Ig1+2, or Ig3+4 of LILRB2 that were expressed on 293T cells. n=3. (FIG.6B) Summary of data from FIG. 6A (FIG. 6C) Summary of Angptl2 bindingabilities of WT and mutant LILRB2. Indicated mutations are described inFIG. 4B. (FIG. 6C) Schematic of the H*G*Y*C motifs in Ig1 (SEQ ID NO:18) and Ig4 (SEQ ID NO: 19) of LILRB2. (FIG. 6D) Summary of Angptl2binding abilities of WT and mutant Ig1+2 LILRB2. (FIG. 6E)Representative flow cytometry plots showing Angptl2 binding to Ig1+2 andmutant LILRB2. (FIG. 6F) Representative flow cytometry plots showingAngptl2 binding to WT and mutant LILRB2. (FIG. 6G) Comparison ofAngptl2, Angptl5, and HLA-G binding abilities of WT and mutant LILRB2.MHC-S indicates HLA-G binding sites; MHC-S1, R59A/Y61A; MHC-S2,W90A/D200A/N202A/Y205A; MHC-S1+2, R59A/Y61A/W90A/D200A/N202A/Y205A.(FIG. 6H) Summary of Angptl2-induced activation of the chimeric receptorreporter system by individual Ig domains, Ig1+2, or Ig3+4 of LILRB2.Indicated reporter cells were treated with 5 μg/ml coated GST-Angptl2 orpolyclonal or monoclonal anti-LILRB2 antibodies. At least threeindependent experiments gave the similar results. (FIG. 6I) Summary ofAngptl2-induced activation of the chimeric receptor reporter system byWT or mutant LILRB2. Reporter cells were treated with 10 μg/ml coatedGST-Angptl2 or polyclonal or monoclonal anti-LILRB2 antibodies. At leastthree independent experiments were performed that gave similar results.

FIG. 7. Interaction of LILRB2 Ig domains with anti-LILRB2 antibodies.Mouse T hybridoma cells were infected with full-length LILRB4 ECD (1-4),individual Ig domains (1st, 2nd, 3rd and 4th Ig domain) and two-Igdomain combinations (1+2 and 3+4). The empty vector was used as negativecontrol. These cells were stained by monoclonal (mAB) or polyclonal(pAB) anti-LILRB2 antibody for flow cytometry analysis.

FIGS. 8A-8C. Mutated residues of LILRB2 in the possible ligand bindinginterface based on the known structure of LILRB2 (SEQ ID NO. 2). Basedon the PDB structure of Ig1-Ig2 domain (PDBID: 2GW5 and 2DYP)(surrounded by solid box in FIG. 8A) and Ig3-Ig4 domain (PDBID: 4LLA)(surrounded by dashed box in FIG. 8A) of human LILRB2, twenty-four largeand hydrophobic residues in the possible ligand binding interface oneach Ig domain were identified for mutagenesis study (underlined in FIG.8A) and generated a series of mutant LILRB2 were generated (FIG. 8B).(FIG. 8C) Summary of Angptl2 binding abilities of WT and mutant LILRB2.

FIG. 9. The FL but not FBN domain of Angptl2 binds to the human cordblood (CB) LILRB2+ cells as determined by flow cytometry analysis. HumanCB mononuclear cells were incubated with indicated FLAG-taggedfull-length, CC domain, or FBN domain of Angptl2 followed by stainingwith anti-FLAGAPC and anti-human LILRB2-PE in a flow cytometry analysis.

FIG. 10. The activation of the LILRB2 chimeric receptor reporter bysoluble or immobilized full-length, CC domain, or FBN domain of Angptl2.Chimeric LILRB2 receptor reporter cells were treated with indicatedsoluble or coated full-length, CC domain, or FBN domain of Angptl2 for48 hrs. PBS was used as control. *, p<0.05; n.s., not significant.

FIGS. 11A-11E. Immobilized anti-LILRB2 antibodies promote theproliferation of human CB cells in vitro. (FIG. 11A) Human CD133+umbilical CB cells were cultured in STF medium with or without sameamounts of coated (25 μg/ml in 50 μl PBS) or soluble (5 μg/ml in 250 μlStemSpan media) anti-LILRB2 pAb. Total cell expansion was assessed after10 days of culture (n=3). (FIG. 11B) Human CD133+ umbilical CB cellswere cultured in STF medium with or without same amounts of coated (25μg/ml in 50 μl PBS) or soluble (5 μg/ml in 250 μl StemSpan media)anti-LILRB2 mAb. Total cell expansion was assessed after 10 days ofculture (n=3). (FIG. 11C) Representative flow cytometric profilesshowing the frequency of CD34+CD90+ cells after 10 days of culture.(FIGS. 11D-11E) Expansion of 250 input equivalent human CB CD133+ cellstreated with or without anti-LILRB2 pAb (FIG. 11D) or mAb (FIG. 11E)were serially plated in CFU medium. Total CFUs were counted after 7 daysin culture. n.s., not significant; *, p<0.05; ***, p<0.001.

FIGS. 12A-12L. Ex vivo expansion of human CB CD133+ cells by anti-LILRB2polyclonal antibody as determined by NSG transplantation. (FIG. 12A)After 10 days of culture in STF medium with or without same amounts ofcoated (25 μg/ml in 50 μl PBS) or soluble (5 μg/ml in 250 μl StemSpanmedia) anti-LILRB2 pAb, expansion of 1×10⁴ input equivalent human CBCD133+ cells were transplanted into NSG mice (n=8). Engraftment of humancells (human CD45+) in peripheral blood at indicated weeks are shown.n.s., not significant; ***, p<0.001. (FIG. 12B) Engraftment of humanCD45/CD71+ in bone marrow of mice described in FIG. 12A at 36 weeks.n.s., not significant; *, p<0.05; n=8. (FIG. 12C) Multilineagecontribution of cultured human umbilical CB CD133+ cells. Shown arerepresentative flow cytometric profiles of bone marrow cells from oneprimary transplanted mouse of each group. Myeloid,CD45/CD71+CD15/CD66b+; lymphoid, CD19/CD20+; hematopoieticstem/progenitor cells, CD19/CD20−CD34+. (FIGS. 12D-12F) Summary ofmultilineage contributions from data shown in FIG. 12C. n.s., notsignificant; *, p<0.05; **, p<0.01; n=8. (FIG. 12G) Engraftment of humanCD45+ cells in peripheral blood of secondarily transplanted mice at 3and 7 weeks post-transplant are shown. n.s., not significant; **,p<0.01; n=3. (FIG. 12H) Engraftments of human cells in bone marrow ofsecondarily transplanted mice at 8 weeks post-transplant are shown.n.s., not significant; *, p<0.05; n=3. (FIG. 12I) Representative flowcytometric profiles showing multilineage contribution of human umbilicalCB CD133+ cells in the bone marrow of secondarily transplanted mice at 8weeks post-transplant. (FIGS. 12J-12L) Summary of multilineagecontributions from data shown in FIG. 12I. n.s., not significant; *,p<0.05; n=3.

FIGS. 13A-13D. Ex vivo expansion of human CB CD34+ cells by anti-LILRB2polyclonal antibody as determined by NSG transplantation. (FIG. 13A)After 10 days culture in STF medium with or without coated or solubleanti-LILRB2 polyclonal antibody, 1×10⁴ input equivalent human CB CD34+cells were transplanted into NSG mice. Engraftments of human CD45/CD71+cells in bone marrow at 8 weeks are shown. n.s., not significant; *,p<0.05; n=8. (FIGS. 13B-13D) Multilineage contribution of cultured humanumbilical CB CD34+ cells. n.s., not significant; *, p<0.05; **, p<0.01;n=8.

FIGS. 14A-14L. Ex vivo expansion of human CB CD133+ cells by anti-LILRB2monoclonal antibody in NSG mice as determined by NSG transplantation.(FIG. 14A) After 10 days culture in STF medium with or without sameamounts of coated (25 μg/ml in 250 μl PBS) or soluble (5 μg/ml in 250 μlStemSpan media) anti-LILRB2 mAb, 1×10⁴ input equivalent human CB CD133+cells were transplanted into NSG mice. Engraftment of human CD45+ inperipheral blood at 3 and 7 weeks are shown. n.s., not significant; *,p<0.05; n=4. (FIG. 14B) Engraftments of human CD45/CD71+ in bone marrowof mice described in FIG. 14A at 8 weeks. n.s., not significant; n=4.(FIG. 14C) Multilineage contribution of cultured human umbilical CBCD133+ cells. Shown are representative flow cytometric profiles of bonemarrow cells from one primary transplanted mouse of each group. (FIGS.14D-14F) Summary of multilineage contributions based on data shown inFIG. 14C. n.s., not significant; n=4. (FIG. 14G) Engraftment of humanCD45+ cells in peripheral blood of secondarily transplanted mice at 3,7, 10, and 30 weeks. n.s., not significant; *, p<0.05; n=3. (FIG. 14H)Engraftment of human cells in bone marrow of secondarily transplantedmice at 30 weeks. n.s., not significant; n=3. (FIG. 141) Representativeflow cytometric profiles showing multilineage contribution of humanumbilical CB CD133+ cells in the bone marrow of secondarily transplantedmice at 8 weeks post-transplant. (FIGS. 14J-14L) Summary of multilineagecontributions based on data from FIG. 141. n.s., not significant; *,p<0.05; n=3.

FIGS. 15A-15I. Net ex vivo expansion of cultured human umbilical CBCD133+ cells as determined by limiting dilution analysis. (FIGS.15A-15B) Numbers of total nucleated cells (FIG. 15A) and CD34+ cells(FIG. 15B) before and after culture with 25 μg/ml coated anti-LILRB2pAB. (C-D) Percentages of donor human CD45+ cells (FIG. 15C) in theperipheral blood at 1 and 2 months and (FIG. 15D) in bone marrow inrecipient NSG mice transplanted with uncultured or expanded cells. (FIG.15E) Net expansion of HSCs as determined by limiting dilution analysis.The numbers of input equivalent cells were used in the calculation.(FIGS. 15F-15I) Comparisons of multilineage repopulation of HSCs beforeand after ex vivo expansion. n.s., not significant; *, p<0.05; **,p<0.01; n=8.

FIG. 16. Clinical grade culture of CB progenitors with Delta1 results inmore rapid neutrophil recovery in a myeloablative double CB transplant(CBT) setting. Individual and median times (solid line) to absoluteneutrophil counts (ANC) of ≥500/μl for patients receiving double unit CBtransplants with two non-manipulated units (“conventional”), one ex vivoexpanded unit and one non-manipulated unit, or the “off-the-shelf”expanded product and a non-manipulated unit. Comparisons made usingtwo-tailed t-test.

FIG. 17. Defining a CD34 Cell Dose for Early Myeloid Engraftment. CD34cell dose versus time to neutrophil engraftment (ANC>500) demonstratingcell doses above which more rapid early myeloid engraftment (before day10) occurs. 6/7 patients with greater than 8 million CD34/kg (blue line)and 5/5 patients with greater than 10 million CD34/kg (red line)demonstrated early engraftment.

FIG. 18. Culture of human CB hematopoietic stem/progenitor cells (HSPC)in the presence of Angptl5 stimulates expansion of in vivo repopulatingcells. Human repopulation in bone marrow of uncultured cells, culturedcells in the absence of Angptl5 (S+T+F) or cells cultured with Angptl5(S+T+F+A5+1). Cell numbers transplanted are represented below conditions(for cultured cells number is progeny of cells generated in culture).

FIG. 19. Culture of CB HSPC in the presence of Delta1 and Angplt5enhances early progenitor and myeloid precursor cell repopulation. CD34+CB HSPC cultured in the presence of Delta1, Angptl5, or the combinationwere transplanted into immunodeficient mice (circles are individualmice, line is median engraftment for group) and progenitor (CD34) andmyeloid (CD33) engraftment assessed at an early time point (2 weekspost-transplant). P-values represent two-tailed t-test.

FIG. 20. CD34-fold expansion of CB HSPC cultured in the presence ofNotch alone, Angptl5 alone, or the combination. CD34+ CB HSPC werecultured for 16 days in the presence of Delta1, Angptl5, or thecombination. CD34 fold expansion (CD34 cells generated/starting numberCD34 cells) was calculated for time points represented (days 7, 10, 16).

FIG. 21. Significantly enhanced early marrow repopulation is seen whenDelta is combined with Angptl5 and cultured in conditions optimized forDelta-mediated expansion.

FIG. 22. Longer-term repopulation is significantly enhanced as well;repopulation is multi-lineage showing significantly enhanced myeloid andlymphoid lineages. Cells did not have significant secondary engraftmentwhen cultured in conditions previously optimized for Delta-mediatedexpansion.

FIGS. 23A and 23B show that culture with Delta and Angptl5 with lowercytokine concentrations results in secondary engraftment previously notseen in Delta expanded cells suggesting maintenance/expansion of alonger-term repopulating cell when Delta is added to ANGPTL5 in theseconditions.

FIG. 24. Culture with Delta and an antibody to the Angptl5 receptor(LILRB2 or CD85) trends towards enhanced early myeloid engraftment ascompared to Delta alone. This trend is present at the highest dose ofCD85 used in this experiment.

FIG. 25. When engraftment was assessed at a longer-term time point (16wks after transplant), engraftment of cells cultured with Delta andantibody to the Angptl5 receptor (LILRB2 or CD85) had greaterengraftment than Delta alone. These cells are able to repopulate bothlymphoid and myeloid lineages.

FIG. 26. CD34+ cord blood hematopoietic stem/progenitor cells werecultured with Delta1 or a combination of Delta1 and anti-LILRB2 antibody(αLILRB2). The progeny of 10,000 starting cells were transplanted intoNSG mice (circles=individual mice, lines=mean engraftment) and myeloidengraftment (y-axis: % CD33+ cells in total marrow) was assessed at 2weeks. P-value represents two tailed t-test.

FIG. 27. CD34+ cord blood hematopoietic stem/progenitor cells werecultured with Delta1 or a combination of Delta1 and anti-LILRB2 antibody(αLILRB2). The progeny of 10,000 starting cells were transplanted intoNSG mice (circles=individual mice, lines=mean engraftment) andprogenitor engraftment (y-axis: % CD34+ cells in total marrow) wasassessed at 10 weeks. P-value represents two tailed t-test.

FIG. 28. Cord blood CD34+ cells were cultured for 4 hrs in non-tissueculture wells coated with retronectin and either i) IgG (human), ii)anti-LILRB2 antibody (αLILRB2), iii) Delta1, or (iv) a combination ofαLILRB2 and Delta1. HES1 expression was assessed by qPCR and normalizedto expression of the β-glucuronidase (GUSB) reference gene. The y-axispresents the data as the fold increase in HES1 expression over the valueobtained for wells coated with retronectin and human IgG.

FIG. 29. Cord blood CD34+ cells were cultured for 4 hrs in non-tissueculture wells coated with retronectin and also coated with either i) IgG(human), ii) a combination of anti-LILRB2 antibody (αLILRB2) and Delta1,or (iii) Delta1, in combination with anti-LILRB2 presented onmicrobeads. HES1 expression was assessed by qPCR and normalized toexpression of the β-glucuronidase (GUSB) reference gene. The y-axispresents the data as the fold increase in HES1 expression over the valueobtained for wells coated with retronectin and human IgG.

DETAILED DESCRIPTION

Hematopoietic precursor cell transplantation represents an importanttherapy due to these cell's capacity to restore blood and immune cellsin transplant recipients. For example, transplantation of precursorcells can be used to treat subjects with inherited immunodeficient orautoimmune diseases and diverse hematopoietic disorders. Precursor celltransplantation can also be used to treat chemotherapy andradiation-treatment patients because prolonged neutropenia andpancytopenia is common following these treatment regimens. As oneexample, and of particular concern, chemotherapeutic treatments for AMLresult in prolonged periods of profound neutropenia with infectiouscomplications still common even in the setting of modern antimicrobialtherapies with mortality rates as high as 20% in adolescent and youngadults. Human bone marrow transplantation methods are also currentlyused as therapies for leukemia, lymphoma, and other life-threateningdiseases.

Delayed myeloid engraftment is a known risk factor for cord blood (CB)transplant (CBT) recipients and is associated with low total nucleatedcell count (TNC) and CD34+ cell doses provided in a single or double CBgraft. The majority of non-relapse mortality in these patients occurswithin the first 100 days post-transplant with infection being the mostcommon cause of death.

In transplantation, high doses of precursor cells are needed to achieverapid and sustained engraftment that is critical for the patient'ssurvival and recovery; this is especially true when CB precursor cellsare used. Although progress toward efficient ex vivo expansion ofprecursor cells has been made, significant improvements in the efficacyand reproducibility of this technology are needed before it can bewidely used.

The present disclosure provides methods for producing immortalized cellpopulations of non-terminally differentiated precursor cells. Inparticular, the present disclosure provides methods of growing precursorcells in culture for a period beyond which the cells would otherwisestop proliferating and/or die, due to senescence and/or undergoingcrisis leading to cell death.

The current disclosure provides improved methods to expand precursorcells through ex vivo expansion. The methods generate more precursorcells more quickly than previously-available methods and the generatedprecursor cells show enhanced early marrow repopulation inimmune-deficient subjects and improved long term repopulation followingtransplant. The methods disclosed herein are based on culturingprecursor cells with an immobilized LILRB2 agonist or an LILRB2 agonistin combination with a Notch agonist.

Preferably, the technique used for expansion is one that has been shownto result in an increase in the number of precursor cells such as HSCe.g., CD34+ cells, in the expanded sample relative to the unexpanded HSCsample. In certain embodiments, the methods result in a 50-, 75-,100-150-, 200-, 250-, 300-, 350-, 400-, 450-, 500-, 1000, 2000-, 3000-,4000-, 5000-fold (or more than) increase in the number of HSC in theexpanded sample, relative to the unexpanded sample. The HSC can bepositive for one or more of CD34, CD43, CD45RO, CD45RA, CD59, CD90,CD109, CD117, CD133, CD166, and HLA DR and/or negative for Lin and/orCD38. In a specific embodiment, the enhanced engraftment can be detectedby detecting an increased percentage of human CD45+ cells in the bonemarrow of mice infused with an aliquot of the expanded sample relativeto mice infused with an aliquot of the unexpanded sample at, e.g., 10days, 3 weeks or 9 weeks post-infusion (see Delaney et al., 2010, NatureMed. 16(2): 232-236). In some embodiments, the methods result in a 50-,75-, 100-, 150-, 200-, 250-, 300-, 350-, 400-, 450-, 500, 1000-, 2000-,3000-, 4000-, 5000-fold (or more than) increase in the number of CD34+HSC in the expanded sample, relative to the unexpanded sample. Cellpopulations are also preferably expanded until a sufficient number ofcells are obtained to provide for at least one infusion into a humansubject.

1.1 LILRB2 and Notch Agonists

The present disclosure contemplates use of an immobilized LILRB2 agonistand/or an LILRB2 agonist in combination with a Notch agonist to expandprecursor cells. An agonist is an agent that promotes, i.e., causes orincreases, activation of LILRB2 and/or Notch pathway function. “LILRB2pathway function” means a function mediated by the LILRB2 signaling(signal transduction) pathway, including inhibition mediated byimmunoreceptor tyrosine-based inhibitory motifs (ITIMs) and/orrecruitment of phosphatases SHP-1, SHP-2, or SHIP. “Notch pathwayfunction” means a function mediated by the Notch signaling (signaltransduction) pathway, including but not limited to nucleartranslocation of the intracellular domain of Notch, nucleartranslocation of RBP-JK or its Drosophila homolog Suppressor ofHairless; activation of bHLH genes of the Enhancer of Split complex,e.g., Mastermind; activation of the HES-1 gene or the KBF2 (also calledCBF1) gene; inhibition of Drosophila neuroblast segregation; and bindingof Notch to a Delta protein, a Jagged/Serrate protein, Fringe, Deltex orRBP-JKI Suppressor of Hairless, or homologs or analogs thereof. Seegenerally the review article by Kopan et al., 2009, Cell 137:216-233 fora discussion of the Notch signal transduction pathway and its effectsupon activation; see also Jarriault et al., 1998, Mol. Cell. Biol.18:7423-7431.

Pathway activation is carried out by exposing a cell to one or moreagonists. The agonists can be but are not limited to soluble molecules,molecules that are recombinantly expressed on a cell-surface, moleculeson a cell monolayer to which the precursor cells are exposed, ormolecules immobilized on a solid phase.

Agonists of the present disclosure include but are not limited toproteins and analogs and derivatives (including fragments) thereof;proteins that are other elements of the LILRB2 or Notch pathway andanalogs and derivatives (including fragments) thereof; activatingantibodies thereto and fragments or other derivatives of such antibodiescontaining the binding region thereof; nucleic acids encoding theproteins and derivatives or analogs; as well as proteins and derivativesand analogs thereof which bind to or otherwise interact with LILRB2 orNotch proteins or other proteins in the LILRB2 or Notch pathways suchthat LILRB2 pathway activity or Notch pathway activity is promoted. Suchagonists include but are not limited to proteins and derivatives thereofcomprising relevant intracellular domains, nucleic acids encoding theforegoing, and proteins comprising the interacting domain of LILRB2 orNotch ligands. These proteins, fragments and derivatives thereof can berecombinantly expressed and isolated or can be chemically synthesized.

Antibodies for use with the methods disclosed herein can include wholeantibodies or binding fragments of an antibody, e.g., Fv, Fab, Fab′,F(ab′)2, Fc, and single chain Fv fragments (ScFv) or any biologicallyeffective fragments of an immunoglobulin that bind specifically to aLILRB2 or Notch motif.

Antibodies or antigen binding fragments include all or a portion ofpolyclonal antibodies, monoclonal antibodies, human antibodies,humanized antibodies, synthetic antibodies, chimeric antibodies,bispecific antibodies, mini bodies, and linear antibodies.

Antibodies from human origin or humanized antibodies have lowered or noimmunogenicity in humans and have a lower number of non-immunogenicepitopes compared to non-human antibodies. Antibodies and theirfragments will generally be selected to have a reduced level or noantigenicity in human subjects.

Antibodies that specifically bind an LILRB2 or Notch motif can beprepared using methods of obtaining monoclonal or polyclonal antibodies,methods of phage display, methods to generate human or humanizedantibodies, or methods using a transgenic animal or plant engineered toproduce antibodies as is known to those of ordinary skill in the art(see, for example, U.S. Pat. Nos. 6,291,161 and 6,291,158). Phagedisplay libraries of partially or fully synthetic antibodies areavailable and can be screened for an antibody or fragment thereof thatcan bind to an LILRB2 or Notch motif. For example, binding domains maybe identified by screening a Fab phage library for Fab fragments thatspecifically bind to a target of interest (see Hoet et al., Nat.Biotechnol. 23:344, 2005). Phage display libraries of human antibodiesare also available. Additionally, traditional strategies for hybridomadevelopment using a target of interest as an immunogen in convenientsystems (e.g., mice, HuMAb Mouse®, TC Mouse™, KM-Mouse®, llamas,chicken, rats, hamsters, rabbits, etc.) can be used to develop bindingdomains. In particular embodiments, antibodies specifically bind anLILRB2 or Notch motif and do not cross react with nonspecific componentsor unrelated targets. Once identified, the amino acid sequence orpolynucleotide sequence coding for the antibody can be isolated and/ordetermined.

In another specific embodiment, the agonist is a cell whichrecombinantly expresses a protein or fragment or derivative thereof,which agonizes LILRB2 or Notch. The cell expresses the agonist in such amanner that it is made available to precursor cells in which LILRB2 orNotch signal transduction is to be activated, e.g., it is secreted,expressed on the cell surface, etc.

In yet another specific embodiment, the agonist is a peptidomimetic orpeptide analog or organic molecule that binds to a member of the LILRB2or Notch signaling pathway. Such an agonist can be identified by thechimeric LILRB2 receptor reporter system described herein and/or bybinding assays selected from those known in the art, for example thecell aggregation assays described in Rebay et al., 1991, Cell 67:687-699and in International Patent Publication No. WO 92119734.

In a preferred embodiment the agonist is a protein including at least afragment of a protein encoded by an LILRB2- or Notch-interacting genewhich mediates binding to a protein or a fragment of LILRB2 or Notch,which fragment contains the region responsible for binding to theagonist protein.

In some embodiments, the agonist is recombinantly expressed from anucleic acid introduced into the precursor cells. In specificembodiments, the recombinantly expressed agonist is a chimeric proteinwhich includes an intracellular domain of a receptor and anextracellular domain of another ligand-binding surface receptor. In suchembodiments, the LILRB2 or Notch pathway can be activated by exposure toa ligand of such another ligand-binding surface receptor. Therecombinantly expressed agonist can be expressed by precursor cells froman inducible promoter. In certain embodiments, the expression of thenucleic acid encoding the agonist is under the control of Cre/Lox systemor FLP/FRT system. In one embodiment, the agonist is flanked by Cresites.

In a specific embodiment, exposure of the cells to an agonist is notdone by incubation with other cells recombinantly expressing a LILRB2 orNotch ligand on the cell surface (although in other embodiments, thismethod can be used), but rather is by exposure to a cell-free ligand,e.g., incubation with a cell-free ligand of LILRB2 or Notch, whichligand is immobilized on the surface of a solid phase, e.g., immobilizedon the surface of a tissue culture dish.

In some embodiments, cells are expanded by culturing the cells with anLILRB2 agonist immobilized on a first solid phase and a Notch agonistimmobilized on a second solid phase, wherein the first and second solidphases are the same.

In some embodiments, cells are expanded by culturing the cells with anLILRB2 agonist immobilized on a first solid phase and a Notch agonistimmobilized on a second solid phase, wherein the second solid phase isnot the first solid phase. In a specific embodiment, the first andsecond solid phases are different types of solid phases, selected fromamong any known in the art, including, but not limited to a culturedish, a culture flask, a culture plate, a bead, a particle, etc. Inspecific embodiments, the first solid phase is a surface of a tissueculture dish or flask, and the second solid phase is a bead, e.g. amagnetic microbead. In other specific embodiments, the first solid phaseis a bead, e.g. a magnetic microbead, and the second solid phase is asurface of a tissue culture dish or flask. In an embodiment where theLILRB2 agonist and Notch agonist are immobilized on different solidphases, the precursor cells can be cultured with the LILRB2 agonist andthe Notch agonist concurrently or sequentially.

1.2 LILRB2 Agonists

The methods disclosed herein include LILRB2 agonists. In particularembodiments, angiopoietins and angiopoietin-like proteins (Angptls)represent exemplary LILRB2 agonists. Until recently, Angptls wereconsidered “orphan ligands” as no receptors were known. In 2012, asubset of the current inventors identified human leukocyteimmunoglobulin-like receptor B2 (LILRB2) and its mouse ortholog pairedIg-like receptor (PirB) as receptors for several Angptls. Zheng et al.,Nature. 2012; 485:656-660. It was also found that LILRB2 and PirB areexpressed by human and mouse HSCs, respectively, and support their exvivo expansion. Zheng et al., Nature. 2012; 485:656-660. LILRB2 sequenceinformation includes: Accession: NP_001265335.2 (SEQ ID NO: 3);Accession: NP_001265334.2 (SEQ ID NO: 4); Accession: NP_001265333.2 (SEQID NO: 5); Accession: NP_001265332.2 (SEQ ID NO: 6); and Accession:AAH36827.1 (SEQ ID NO: 7).

Several Angptls support the activity of precursor cells, such as HSCs invitro and in vivo. For example, several Angptls inhibit differentiationand promote repopulation of HSCs in vitro and in vivo. Zheng et al.,Cell Stem Cell. 2011; 9:119-130; Zheng et al., Blood. 2011; 117:470-479;Zhang and Lodish Curr Opin Hematol. 2008; 15:307-311; and Zhang et al.,Blood. 2008; 111:3415-3423. LILRB2 and PirB are also required forleukemia development as they inhibit differentiation and promoteself-renewal of leukemic progenitors. Zheng et al., Nature. 2012;485:656-660. It was further demonstrated that the binding of Angptls toLILRB2/PirB induces activation of SHP-2 and CAMKs, both types of factorsknown to be critical for supporting the activity of HSCs Kitsos et al.,J Biol Chem. 2005; 280:33101-33108; Chan et al., Exp Hematol. 2006;34:1230-1239.

LILRB2 receptors contain ITIMs in their intracellular domains and areclassified as inhibitory receptors because ITIM motifs can recruitphosphatases SHP-1, SHP-2, or SHIP to negatively regulate cellactivation. Takai et al., J Biomed Biotechnol. 2011; 2011:275302; Daeronet al., Immunol Rev. 2008; 224:11-43. An important question is howAngptl binding leads to the activation of LILRB2. In Example 1, themolecular basis for the interaction between Angptls and LILRB2 isdescribed. It is shown that mammalian-expressed Angptl2 exists as HMWspecies, which is needed for activation of LILRB2 and subsequentdownstream signaling. A novel motif in the first and fourth Ig domainsof LILRB2 that is critical to the Angptl2 binding was also identified.Moreover, that the binding of Angptl2 to LILRB2 is more potent and notcompletely overlapped with the binding of another ligand HLA-G is shown.Based on the new understanding of the Angptl/LILRB2 interaction, aserum-free culture containing defined cytokines and immobilizedantiLILRB2 antibodies that supports a stable and reproducible ex vivoexpansion of repopulating human CB precursor cells was developed.

Based on the foregoing, LILRB2 agonists of the current disclosure canparticularly include high molecular weight agonists (above 200 kD; above210 kD; above 215 kD; above 220 kD; above 225 kD; above 230 kD; above235 kD; above 240 kD; above 245 kD; above 250 kD; above 255 kD; above260 kD; above 265 kD; above 270 kD; above 275 kD; above 280 kD; above285 kD; above 290 kD; above 295 kD; or above 300 kD) including highmolecular weight Angptls. LILRB2 agonists can also include multimerizedLILRB2 agonists, including mulitmerized Angptls.

In particular embodiments, LILRB2 agonists bind a motif in the Ig1domain of LILRB2, a motif in the Ig2 domain of LILRB2, a motif in theIg3 domain of LILRB2, and/or a motif in the Ig4 domain of LILRB2. Inmore particular embodiments, the LILRB2 agonists bind motifs in the Ig1and Ig4 domains of LILRB2. In more particular embodiments, the LILRB2agonists bind amino acids at positions 92-100 of the Ig1 domain. In moreparticular embodiments, the LILRB2 agonists bind amino acids atpositions 390-396 of the Ig4 domain. In more particular embodiments, theLILRB2 agonists bind amino acids at positions 92-100 of the Ig1 domainand amino acids at positions 390-396 of the Ig4 domain. In moreparticular embodiments, the LILRB2 agonists bind amino acids atpositions 94, 95 and 96 of the Ig1 domain and amino acids at positions392 and 394 of the Ig4 domain.

Within the current disclosure, Angptls can be any member of a family ofsecreted glycosylated proteins that are similar in structure toangiopoietins (Oike et al., Int. J. Hematol. 80:21-8 (2004)). Similar toangiopoietins, Angptls contain an N-terminal CC domain and a C-terminalFBN-like domain. Unlike angiopoietins, Angptls do not bind to thetyrosine kinase receptor Tie2. Angptls include Angptls 2, 3, 4, 5, 6,and 7. Angptls also include microfibrillar-associated glycoprotein 4(Mfap4), and analogs and equivalents thereof. Angptl2 has been describedby Kim, et al. 1999, J Biol Chem 274, 26523-8). In addition, Angptls areavailable commercially (R&D Systems, Abnova Corp).

Exemplary Angptls are provided, for example in GenBank as AccessionNumber AAH12368 (human Angptl2 precursor; SEQ ID NO: 8) Accession NumberAAH58287 (human Angptl3 precursor; SEQ ID NO: 9) Accession NumberAAH23647 (human Angptl4; SEQ ID NO: 10) and Accession Number AAH49170(human Angptl5; SEQ ID NO: 11). An exemplary sequence for Angptl7 isfound in GenBank Accession No. AAH01881 (SEQ ID NO: 12). An exemplarysequence for Mfap4 is found in GenBank Accession No. NP002395 (SEQ IDNO: 13).

Suitable equivalents for Angptls include proteins and polypeptideshaving similar biological activity to these factors as wild-type orpurified Angptls (e.g., recombinantly produced). Suitable analogs ofAngptls include fragments retaining the desired activity and relatedmolecules. One preferred analog is a fragment of the Angptl containingthe CC domain, for example, the CC domain of Angptl2. Another analog isthe FBN-like domain. Fragments of Angptls such as the CC domain and theFBN-like domain may be easier to express and to purify compared tofull-length protein. Molecules capable of binding the correspondingAngptl receptor and initiating one or more biological actions associatedwith Angptl binding to its receptor are also within the scope of thedisclosure.

Antibodies to the LILRB2 receptor can also be used. Exemplarycommercially available antibodies include, anti-LILRB2 polyclonalantibody (pAb, #BAF2078, R&D systems) and anti-LILRB2 monoclonalantibody (mAb, #16-5149-85, eBioscience).

Exemplary production methods of LILRB2 agonists are described in, forexample, U.S. Patent Publication Nos. 2014/0017784 and 2011/0196343.

In certain embodiments, to determine whether an LILRB2 binding protein,e.g., an anti-LILRB2 antibody, is an LILRB2 agonist, precursor cells,e.g., hematopoietic stem cells or hematopoietic progenitor cells, arecultured in the presence of the LILRB2 binding protein and then testedfor increased Hes1 expression levels (relative to precursor cellscultured in the presence of a control molecule not having LILRB2 agonistactivity), e.g., by q-PCR, wherein increased Hes1 expression levels inthe cells cultured in the presence of the LILRB2 binding proteinindicates that the LILRB2 binding protein is an LILRB2 agonist. In otherembodiments, to determine whether an LILRB2 binding protein, e.g., ananti-LILRB2 antibody, is an LILRB2 agonist, precursor cells, e.g.,hematopoietic stem cells or hematopoietic progenitor cells, are culturedin the presence of the LILRB2 binding protein and then injected into NSGmice, wherein increased engraftment of the cells cultured in thepresence of the LILRB2 binding protein in NSG mice (relative toprecursor cells cultured in the presence of a control molecule nothaving LILRB2 agonist activity) indicates that the LILRB2 bindingprotein is an LILRB2 agonist.

1.3 Notch Agonists

The current disclosure also describes stable and reproducible ex vivoexpansion of precursor cells using a combination of Angptl agonists andNotch agonists.

Members of the Notch family encode large transmembrane proteins thatplay central roles in cell-cell interactions and cell-fate decisionsduring early development in a number of invertebrate systems (Simpson,1995, Nature 375:736-7; Artavanis-Tsakonis et al., 1995, Science.268:225-232; Simpson, 1998, Semin. Cell Dev. Biol. 9:581-2; Go et al.,1998, Development. 125:2031-2040; Artavanis-Tsakonas and Simpson, 1991,Trends Genet. 7:403-408). The Notch receptor is part of a highlyconserved pathway that enables a variety of cell types to choose betweenalternative differentiation pathways based on those taken by immediatelyneighboring cells. This receptor appears to act through an undefinedcommon step that controls the progression of uncommitted cells towardthe differentiated state by inhibiting their competence to adopt one oftwo alternative fates, thereby allowing the cell either to delaydifferentiation, or in the presence of the appropriate developmentalsignal, to commit to differentiate along the non-inhibited pathway.

Genetic and molecular studies have led to the identification of a groupof genes which define distinct elements of the Notch signaling pathway.While the identification of these various elements has come fromDrosophila using genetic tools, initial guide, subsequent analyses haveled to the identification of homologous proteins in vertebrate speciesincluding humans. The molecular relationships between the known Notchpathway elements as well as their subcellular localization are depictedin Artavanis-Tsakonas et al., 1995, Science 268:225-232;Artavanis-Tsakonas et al., 1999, Science 284:770-776; and in Kopan etal., 2009, Cell 137:216-233. Proteins of the Delta family and proteinsof the Serrate (including Jagged, the mammalian homolog of Serrate)family are extracellular ligands of Notch. The portion of Delta andSerrate responsible for binding to Notch is called the DSL domain, whichdomain is located in the extracellular domain of the protein. Epidermalgrowth factor-like repeats (ELRs) 11 and 12 in the extracellular domainof Notch are responsible for binding to Delta, Serrate and Jagged. SeeArtavanis-Tsakonas et al., 1995, Science 268:225-232 and Kopan et al.,2009, Cell 137:216-233. Exemplary sequences relevant to Notch signalinginclude Accession Number: P46531.4 (SEQ ID NO: 14); Accession Number:AAG09716.1 (SEQ ID NO: 15); Accession Number: 2KB9_A (SEQ ID NO: 16) andAccession Number: 2VJ2_B (SEQ ID NO: 17).

The present disclosure contemplates use of a Notch agonist. Contemplatedfor use in the present disclosure are any of the Notch agonistsdisclosed in U.S. Pat. No. 7,399,633, or any other Notch agonists knownin the art.

Notch agonists include but are not limited to Notch proteins andderivatives thereof comprising the intracellular domain, Notch nucleicacids encoding the foregoing, and proteins comprising theNotch-interacting domain of Notch ligands (e.g., the extracellulardomain of Delta or Serrate). Other agonists include but are not limitedto RBP JKI Suppressor of Hairless or Deltex. Fringe can be used toenhance Notch activity, for example in conjunction with Delta protein.These proteins, fragments and derivatives thereof can be recombinantlyexpressed and isolated or can be chemically synthesized.

In a preferred embodiment the agonist is a protein including at least afragment of a protein encoded by a Notch-interacting gene which mediatesbinding to a Notch protein or a fragment of Notch, which fragment ofNotch contains the region of Notch responsible for binding to theagonist protein, e.g., epidermal growth factor-like repeats 11 and 12 ofNotch. Notch interacting genes mean the genes Notch, Delta, Serrate,Jagged, RBPJK, Suppressor of Hairless and Deltex, as well as othermembers of the Delta/Serrate family or Deltex family which may beidentified by virtue of sequence homology or genetic interaction andmore generally, members of the “Notch cascade” or the “Notch group” ofgenes, which are identified by molecular interactions (e.g., binding invitro, or genetic interactions (as depicted phenotypically, e.g., inDrosophila). Exemplary fragments of Notch-binding proteins containingthe region responsible for binding to Notch are described in U.S. Pat.Nos. 5,648,464; 5,849,869; and 5,856,441. The Notch agonists utilized bythe methods of the disclosure can be obtained commercially, produced byrecombinant expression, or chemically synthesized.

In a specific embodiment, the Notch agonist is a dominant active mutantof a Notch protein (e.g., a Notch receptor lacking the extracellular,ligand binding domain). In another embodiment, the Notch agonist is nota dominant active mutant of a Notch protein.

In some embodiments, the Notch agonist is recombinantly expressed from anucleic acid introduced into the precursor cell. Methods that can beused for recombinantly expressing a Notch agonist are described in sec.5.3 of U.S. Pat. No. 7,399,633. In particular embodiments, the Notchagonist is a Notch protein (e.g., human or murine Notch-1, Notch-2,Notch-3 or Notch-4) including the intracellular domain of the Notchprotein expressed recombinantly in precursor cells. In specificembodiments, the recombinantly expressed Notch agonist is a chimericNotch protein which includes the intracellular domain of Notch receptorand the extracellular domain of another ligand-binding surface receptor(e.g., the EGF receptor). In such embodiments, the Notch pathway can beactivated by exposure to a ligand of such another ligand-binding surfacereceptor (e.g., EGF). The recombinantly expressed Notch agonist can beexpressed by precursor cells from an inducible promoter. In certainembodiments, the expression of the nucleic acid encoding the Notchagonist is under the control of Cre/Lox system or FLP/FRT system. In oneembodiment, the Notch agonist is flanked by Cre sites.

In another specific embodiment, and as described in U.S. Pat. No.5,780,300 to Artavanis-Tsakonas et al., Notch agonists include reagentsthat promote or activate cellular processes that mediate the maturationor processing steps required for the activation of Notch or a member ofthe Notch signaling pathway, such as the turin-like convertase requiredfor Notch processing, Kuzbanian, the metalloprotease-disintegrin (ADAM)thought to be required for the activation of the Notch pathway upstreamor parallel to Notch (Schlondorfiand Blobel, 1999, J. Cell Sci.112:3603-3617), or, more generally, cellular trafficking and processingproteins such as the rab family of GTPases required for movement betweencellular compartments (for a review on Rab GTPases, see Olkkonen andStenmark, 1997, Int. Rev. Cytol. 176:1-85). The agonist can be anymolecule that increases the activity of one of the above processes, suchas a nucleic acid encoding a turin, Kuzbanian or rab protein, or afragment or derivative or dominant active mutant thereof: or apeptidomimetic or peptide analog or organic molecule that binds to andactivates the function of the above proteins.

U.S. Pat. No. 5,780,300 further discloses classes of Notch agonistmolecules (and methods of their identification) which can be used toactivate the Notch pathway in the practice of the present disclosure,for example molecules that trigger the dissociation of the Notch ankyrinrepeats with RBP-JK, thereby promoting the translocation of RBP-JK fromthe cytoplasm to the nucleus.

Exemplary Notch agonists are the extracellular binding ligands Delta andSerrate (e.g., Jagged) which bind to the extracellular domain of Notchand activate Notch signal transduction, or a fragment (e.g., theextracellular domain) of Delta or Serrate (e.g., Jagged) that binds tothe extracellular domain of Notch and activates Notch signaltransduction. Nucleic acid and amino acid sequences of Delta familymembers and Serrate family members (e.g., Jagged family members) havebeen isolated from several species, including human, are known in theart, and are disclosed in International Patent Publication Nos. WO93/12141, WO 96/27610, WO 97/01571, Gray et al., 1999, Am. J. Path.154:785-794. Jagged is a mammalian homologue of Serrate. As used in thisapplication, Serrate shall encompass Jagged unless the context indicatesotherwise. In one embodiment, the Notch agonist is Delta^(extIgG), whichis a fragment of a Delta protein consisting of the extracellular domainof the protein fused to the Fc portion of IgG.

In certain embodiments, the Notch agonist is an anti-Notch antibody orantigen-binding fragment thereof. In specific embodiments, the Notchagonist is an anti-Notch-1 antibody or antigen-binding fragment thereof.In more specific embodiments, the Notch agonist is the anti-Notch-1HMN1-519 antibody (commercially available from Biolegend, San Diego,Calif.). In other specific embodiments, the Notch agonist is ananti-Notch-2 antibody or antigen-binding fragment thereof. In morespecific embodiments, the Notch agonist is the anti-Notch-2 HMN2-25antibody (commercially available from Biolegend, San Diego, Calif.). Inother specific embodiments, the Notch agonist is a combination of ananti-Notch-1 antibody and an anti-Notch-2 antibody.

Sequence information provided by public databases can be used toidentify nucleic acid sequences encoding proteins disclosed herein andvice versa. Variants of the sequences disclosed and referenced hereinare also included. Variants of proteins can include those having one ormore conservative amino acid substitutions. As used herein, a“conservative substitution” involves a substitution found in one of thefollowing conservative substitutions groups: Group 1: Alanine (Ala orA), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3:Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg orR), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile orI), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); andGroup 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trpor W).

Additionally, amino acids can be grouped into conservative substitutiongroups by similar function or chemical structure or composition (e.g.,acidic, basic, aliphatic, aromatic, sulfur-containing). For example, analiphatic grouping may include, for purposes of substitution, Gly, Ala,Val, Leu, and Ile. Other groups containing amino acids that areconsidered conservative substitutions for one another include:sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn,and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser,Thr, Pro, and Gly; polar, negatively charged residues and their amides:Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg,and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, andCys; and large aromatic residues: Phe, Tyr, and Trp. Additionalinformation is found in Creighton (1984) Proteins, W.H. Freeman andCompany.

Variants of the protein sequences disclosed or referenced herein alsoinclude sequences with at least 70% sequence identity, at least 80%sequence identity, at least 85% sequence, at least 90% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, or at least 99% sequence identity to the protein sequencesdisclosed or referenced herein.

“% sequence identity” refers to a relationship between two or moresequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenproteins as determined by the match between strings of such sequences.“Identity” (often referred to as “similarity”) can be readily calculatedby known methods, including (but not limited to) those described in:Computational Molecular Biology (Lesk, A. M., ed.) Oxford UniversityPress, N Y (1988); Biocomputing: Informatics and Genome Projects (Smith,D. W., ed.) Academic Press, N Y (1994); Computer Analysis of SequenceData, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.)Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. andDevereux, J., eds.) Oxford University Press, NY (1992). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR, Inc., Madison, Wis.). Multiple alignment of thesequences can also be performed using the Clustal method of alignment(Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters(GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also includethe GCG suite of programs (Wisconsin Package Version 9.0, GeneticsComputer Group (GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul,et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc.,Madison, Wis.); and the FASTA program incorporating the Smith-Watermanalgorithm (Pearson, Comput. Methods Genome Res., [Proc. Int.Symp.](1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor.Publisher: Plenum, New York, N.Y. Within the context of this disclosureit will be understood that where sequence analysis software is used foranalysis, the results of the analysis are based on the “default values”of the program referenced. As used herein “default values” will mean anyset of values or parameters which originally load with the software whenfirst initialized.

1.4 Immobilized/Solid Support

In some embodiments, during the culturing step, precursor cells arecultured in the presence of immobilized LILRB2 and/or Notch agonists, inparticular embodiments the extracellular domains of an agonist, and infurther particular embodiments, fused to a fusion partner. In specificembodiments, during the culturing step, precursor cells are cultured ona solid phase coated with LILRB2 and/or Notch agonists.

In certain embodiments, the isolated precursor cells are expanded in thepresence of a fibronectin or a fragment thereof (e.g., CH-296; (Dao etal., 1998, Blood 92(12):4612-21)) or RetroNectin® (a recombinant humanfibronectin fragment; Clontech Laboratories, Inc., Madison, Wis.)). Incertain embodiments, fibronectin is excluded from the tissue culturedishes or is replaced by another extracellular matrix protein. See alsoU.S. Pat. No. 7,399,633 to Bernstein et al. for additional exemplaryculture conditions for precursor cell expansion. For example, theisolated precursor cells can be expanded in the presence of animmobilized fibronectin or a fragment thereof (e.g., immobilized on thesame solid phase as LILRB2 and/or Notch agonist), or immobilized on asolid phase that is different from the solid phase on which the LILRB2and/or Notch agonist is immobilized.

1.5 Growth Factors & Other Culture Components

In some embodiments, precursor cells are expanded in the presence of oneor more growth factors, two or more growth factors, three or more growthfactors, or four or more growth factors (e.g., in a fluid medium).

In some embodiments, the amount or concentration of growth factorssuitable for expanding precursor cells of the present disclosure is theamount or concentration effective to promote proliferation of HSC butsubstantially no differentiation of HSC.

Exposing precursor cells to one or more growth factors can be done priorto, concurrently with, or following exposure of the cells to a LILRB2and/or Notch agonist. In some embodiments, precursor cells are exposedto one or more growth factors for at least a portion of the time or theminimal culture time, most preferably the majority or all of the time,that precursor cells are exposed to a LILRB2 and/or Notch agonist. Theminimal culture time is the amount of time at which the cell would dieor stop proliferating in the absence of LILRB2 and/or Notch agonist andthe growth factors (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or 25 weeks). In specificembodiments, the minimal culture time is from 3 to 4 weeks.

In specific exemplary embodiments, the growth factors present in theexpansion medium include one or more of the following growth factors:stem cell factor (SCF; also known as the c-kit ligand or mast cellgrowth factor), Flt-3 ligand (Flt-3L), interleukin-6 (IL-6),interleukin-3 (IL-3), interleukin-7 (IL-7), interleukin-11 (IL-11),thrombopoietin (TPO), granulocyte-macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), insulin growthfactor-2 (IFG-2), and fibroblast growth factor-1 (FGF-1).

In some embodiments, the growth factors present in the expansion mediuminclude one or more of the following growth factors: IL-I, IL-3, IL-6,IL-11, G-CSF, GM-CSF, SCF, FIT3-L, TPO, erythropoietin and analogsthereof (wherein the analogs include any structural variants of thegrowth factors having the biological activity of the naturally occurringgrowth factor and cytokine receptor agonists, e.g., agonist antibodyagainst the TPO receptor such as VB22B sc(Fv)2 described in WO20071145227) (see page 13 of U.S. Patent Publication No. 2010/0183564).In one embodiment, SCF, Flt3-L and TPO are used in the expansion methodsprovided herein. In another embodiment, IL-6, SCF, Flt3-L and TPO areused in the expansion methods provided herein. In some embodiments, oneor more growth factors are used in a serum-free medium.

The amount or concentration of growth factors suitable for expandingprecursor cells of the present disclosure will depend on the activity ofthe growth factor preparation, and the species correspondence betweenthe growth factors and precursor cells, etc. The amount of growthfactors can be in the range of 5-1000 ng/ml. Generally, when the growthfactor(s) and precursor cells are of the same species, the total amountof growth factor in the culture medium ranges from 1 ng/ml to 100 μg/ml,more preferably from 5 ng/ml to 1 μg/ml, and most preferably from about5 ng/ml to 250 ng/ml.

In certain embodiments, the foregoing growth factors are present in theculture condition for expanding precursor cells at the followingconcentrations: 25-300 ng/ml SCF, 25-300 ng/ml Flt-3 ligand, 25-100ng/ml TPO, 25-100 ng/ml IL-6 and 10 ng/ml IL-3. In more specificembodiments, 50, 100 or 200 ng/ml SCF, 50, 100 or 200 ng/ml of Flt-3ligand, 50 or 100 ng/ml TPO, 50 or 100 ng/ml IL-6 and about 10 ng/mlIL-3 can be used.

In one embodiment, precursor cells are expanded by exposing precursorcells to an LILRB2 agonist and 50 ng/ml SCF; 10 ng/ml TPO; and 50 ng/mlFLT3-ligand. In one embodiment, precursor cells are expanded by exposingprecursor cells to an LILRB2 agonist and 50 ng/ml SCF; 10 ng/ml TPO; and50 ng/ml FLT3-ligand in StemSpan media (Stemcell Technologies, Inc.). Inanother embodiment, precursor cells are expanded by exposing precursorcells to an LILRB2 agonist and 50 ng/ml SCF; 10 ng/ml TPO; and 50 ng/mlFLT3-ligand for 10 days.

In further embodiments, precursor cells are expanded by exposingprecursor cells to an LILRB2 agonist, a Notch agonist and 50 ng/ml SCF;50 ng/ml Flt-3 ligand; 50 ng/ml interleukin-6 (IL-6); 50 ng/ml TPO; 20ng/ml FGF1; 10 ng/ml interleukin-3 (IL-3); and 10 μg/ml heparin. In oneembodiment, precursor cells are expanded by exposing precursor cells toan LILRB2 agonist, a Notch agonist and 50 ng/ml SCF; 50 ng/ml Flt-3ligand; 50 ng/ml interleukin-6 (IL-6); 50 ng/ml TPO; 20 ng/ml FGF1; 10ng/ml interleukin-3 (IL-3); and 10 μg/ml heparin in StemSpan media(Stemcell Technologies, Inc.).

Exposing precursor cells to a Notch agonist can be done prior to,concurrently with, or following exposure of the cells to an LILRB2agonist. In one embodiment, precursor cells are exposed to both anLILRB2 agonist and a Notch agonist for the entire period of ex vivoexpansion of precursor cells. In some embodiments, precursor cells areexposed to both an LILRB2 agonist and a Notch agonist for more than 80%,85%, 90%, 95%, 98%, or 99% of the period of ex vivo expansion ofprecursor cells. In another embodiment, precursor cells are exposed toan LILRB2 agonist and a Notch agonist for less than the entire period ofex vivo expansion of precursor cells. In yet another embodiment,precursor cells are exposed to an LILRB2 agonist for the entire periodof ex vivo expansion of precursor cells, but are exposed to a Notchagonist for less than the entire period of ex vivo expansion (e.g., forless than 100%, 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 60%, or 50% ofthe ex vivo expansion period). Alternatively, precursor cells areexposed to a Notch agonist for the entire period of ex vivo expansion ofprecursor cells, but are exposed to an LILRB2 agonist for less than theentire period of ex vivo expansion (e.g., for less than 100%, 99%, 98%,95%, 90%, 85%, 80%, 75%, 70%, 60%, or 50% of the ex vivo expansionperiod).

Culturing precursor cells can take place under any suitable culturemedium/conditions described in U.S. Patent Publication No. 2004/0067583,U.S. Pat. No. 7,399,633, or U.S. Patent Publication No. 2010/0183564 oras is known in the art (see, e.g., Freshney Culture of Animal Cells,Wiley-Liss, Inc., New York, N.Y. (1994)). The time in culture is a timesufficient to produce an expanded precursor cell population. Forexample, precursor cells can be cultured in a serum-free medium in thepresence of an LILRB2 agonist and/or a Notch agonist, and, optionally,one or more growth factors or cytokines for 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, or 35 days; or, preferably, for at least 10 or at least 15 daysor at least 16 days. Optionally, at any point during the culturingperiod, the culture medium can be replaced with fresh medium or freshmedium can be added. In one embodiment, the fresh culture medium isadded every 3 or 4 days.

In other embodiments, precursor cells are cultured for 1 week, 2 weeks,3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks or 10weeks; or, preferably, the precursor cells are cultured for at least 3or 4 weeks (in the presence of the combination of an LILRB2 agonistand/or a Notch agonist, and, optionally, one or more growth factors). Inyet other embodiments, precursor cells are cultured for less than 4weeks (in the presence of an LILRB2 agonist and/or a Notch agonist, and,optionally, one or more growth factors). In yet other embodiments,precursor cells are cultured for more than 10 weeks, e.g., 12, 15, 18,20 or 25 weeks (in the presence of an LILRB2 agonist and/or a Notchagonist, and, optionally, one or more growth factors).

1.6 Source, Collection, Isolation and Treatment of Precursor Cells forExpansion

The present disclosure provides methods for immortalizing and optionallydifferentiating precursor cells, by circumventing or delaying the entryof the precursor cells into cell cycle arrest or into a nonreplicativephase. Precursor cells for immortalization according to the disclosureare non-terminally-differentiated cells and can be from any species,including but not limited to human, animal, plant, mammal, primate,mouse, rat, dog, cat, horse, cow, fowl, insect, Drosophila, and C.elegans. Most preferably, the precursor cells are vertebrate, morepreferably mammalian, and most preferably human. In a preferredembodiment, the precursor cells are have not gone through a “crisis” or“senescence” phase resulting in cell line characteristics (e.g.transformation resulting in a stable phenotypic change (see Freshney,1994, In “Culture of Animal Cells—A manual of Basic Technique,” 3.sup.rdEdition at p. 12, John Wiley & Sons, Inc.). In a preferred embodiment,the precursor cells are primary cells. The term “primary cells”indicates that the cells are have not been through a subculturefollowing their explanation from a tissue source, such as a mammaliansubject.

Generally, though not necessarily, the precursor cells are pluripotentstem cells or multipotent progenitor cells. In one embodiment, theprecursor cells are stem cells. In another embodiment, the precursorcells are progenitor cells. The precursor cells can be isolated from acell population, if desired, before or after immortalization.

In a specific embodiment, the precursor cells are hematopoietic stemcells. In a specific embodiment, the precursor cells are hematopoieticprogenitor cells.

In a specific embodiment, the precursor cells are a population of cellsenriched for hematopoietic stem cells. In another embodiment, theprecursor cells are a population of cells enriched for hematopoieticstem and progenitor cells.

Sources of precursor cells include but are not limited to: umbilical CB,placental blood, peripheral blood (e.g., mobilized peripheral blood),bone marrow (e.g., from femurs, hips, ribs, sternum and other bones),embryonic cells (including embryonic stem cells and hematopoeiticprecursors of HSC derived from embryonic stem cells, induced pluripotentstem cells or HSC or hematopoietic precursors derived by reprogramming(see Gazit et al., Stem Cell Reports, Vol. 1, 266-280, 2013),aortal-gonadal-mesonephros derived cells, lymph, liver (e.g., fetalliver), thymus, and spleen. Sources of precursor cells further includefetal blood, neonatal blood (from an infant in the first 28 days afterbirth), blood from an infant under 12 months of age, blood from atoddler between 1 year and 3 years of age, blood form a child between 3and 18 years of age, and adult blood (i.e., derived from a subject whois older than 18 years of age). As will be understood by one of ordinaryskill in the art, all collected samples can be screened for undesirablecomponents and discarded, treated or used according to accepted currentstandards.

Umbilical CB and/or placental blood can be obtained by any method knownin the art. The use of cord or placental blood as a source of CB stemcells provides numerous advantages, including that the cord andplacental blood can be obtained easily and without trauma to the donor.See, e.g., U.S. Pat. No. 5,004,681 for a discussion of collecting cordand placental blood at the birth of a human. In one embodiment CBcollection is performed by the method disclosed in U.S. Pat. No.7,147,626.

Collections should be made under sterile conditions. Immediately uponcollection, cord or placental blood should be mixed with ananticoagulant. Such an anticoagulant can be any known in the art,including but not limited to CPO (citrate-phosphate-dextrose), ACD (acidcitrate-dextrose), Alsever's solution (Alsever et al., 1941, N.Y. St. J.Med. 41:126), De Gowin's Solution (De Gowin, et al., 1940, J. Am. Med.Ass. 114:850), Edglugate-Mg (Smith, et al., 1959, J. Thorac. Cardiovasc.Surg. 38:573), Rous-Tumer Solution (Rous and Turner, 1916, J. Exp. Med.23:219), other glucose mixtures, heparin, ethyl biscoumacetate, etc.See, generally, Hum, 1968, Storage of Blood, Academic Press, New York,pp. 26-160. In particular embodiments, ACD can be used.

The CB can preferably be obtained by direct drainage from the cordand/or by needle aspiration from the delivered placenta at the root andat distended veins. See, generally, U.S. Pat. No. 5,004,681. Preferably,the collected human CB and/or placental blood is free of contamination.

In certain embodiments, HSC are obtained from the fetal blood from thefetal circulation at the placental root with the use of needle guidedultrasound, by placentocentisis, or by fetoscopy as described in sec.5.4.5 of U.S. Pat. No. 7,399,633. In specific embodiments, HSC areobtained from Wharton's jelly as described in sec. 5.4.5 of U.S. Pat.No. 7,399,633.

Peripheral blood is preferably mobilized prior to its collection.Peripheral blood can be mobilized by any method known in the art.Peripheral blood can be mobilized by treating the subject from whomprecursor cells are to be collected with any agent(s), described hereinor known in the art, that increase the number of precursor cellscirculating in the peripheral blood of a subject. For example, in someembodiments, peripheral blood is mobilized by treating the subject fromwhom precursor cells are to be collected with one or more cytokines orgrowth factors (e.g., G-CSF, kit ligand (KL), IL-I, IL-7, IL-8, IL-11,Flt3 ligand, SCF, thrombopoietin, or GM-CSF (such as sargramostim)).Different types of G-CSF that can be used in the methods formobilization of peripheral blood include, without limitation, filgrastimand longer acting G-CSF-pegfilgrastim. In certain embodiments,peripheral blood is mobilized by treating the subject from whomprecursor cells are to be collected with one or more chemokines (e.g.,macrophage inflammatory protein-I a (MIP1a/CCL3)), chemokine receptorligands (e.g., chemokine receptor 2 ligands GROP and GROPM), chemokinereceptor analogs (e.g., stromal cell derived factor-1a (SDF-1a) peptideanalogs such as CTCE-0021, CTCE-0214, or SDF-1a such as Met-SDF-Ip), orchemokine receptor antagonists (e.g., chemokine (C-X-C motif) receptor 4(CXCR4) antagonists such as AMD3100). In some embodiments, peripheralblood is mobilized by treating the subject from whom precursor cells areto be collected with one or more anti-integrin signaling agents (e.g.,function blocking anti-very late antigen 4 (VLA-4) antibody, oranti-vascular cell adhesion molecule 1 (VCAM-1)). In other embodiments,peripheral blood is mobilized by treating the subject from whomprecursor cells are to be collected with one or more cytotoxic drugssuch as cyclophosphamid, etoposide or paclitaxel. In particularembodiments, peripheral blood can be mobilized by administering to asubject one or more of the agents listed above for a certain period oftime. For example, the subject can be treated with one or more agents(e.g., G-CSF) via injection (e.g., subcutaneous, intravenous orintraperitoneal), once daily or twice daily, for 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 days prior to collection of precursor cells. Inspecific embodiments, precursor cells are collected within 1, 2, 3, 4,5, 6, 7, 8, 12, 14, 16, 18, 20 or 24 hours after the last dose of anagent used for mobilization of peripheral blood. In some embodiments,peripheral blood is mobilized by treating the subject from whomprecursor cells are to be collected with two or more different types ofagents described above or known in the art, such as a growth factor(e.g., G-CSF) and a chemokine receptor antagonist (e.g., CXCR4 receptorantagonist such as AMD3100), or a growth factor (e.g., G-CSF or KL) andan anti-integrin agent (e.g., function blocking VLA-4 antibody). Inparticular embodiments, different types of mobilizing agents areadministered concurrently or sequentially. Methods of mobilization ofperipheral blood are known in the art (see, e.g., Craddock et al., 1997,Blood 90(12):4779-4788; Jin et al., 2008, Journal of TranslationalMedicine 6:39; Pelus, 2008, Curr. Opin. Hematol. 15(4):285-292;Papayannopoulou et al., 1998, Blood 91(7):2231-2239; Tricot et al.,2008, Haematologica 93(11):1739-1742; Weaver et al., 2001, Bone MarrowTransplantation 27(2):S23-S29).

Precursor cells from bone marrow can be obtained, e.g., directly frombone marrow from the posterior iliac crest by needle aspiration (see,e.g., Kodo et al., 1984, J. Clin Invest. 73:1377-1384), or from theblood following pre-treatment with cytokines (such as G-CSF) that inducecells to be released from the bone marrow compartment. Precursor cellsfrom peripheral blood can be collected from the blood through a syringeor catheter inserted into a subject's vein. For example, the peripheralblood can be collected using an apheresis machine. Blood flows from thevein through the catheter into an apheresis machine, which separates theprecursor cells from the rest of the blood and then returns the blood tothe subject's body. Apheresis can be performed for several days (e.g., 1to 5 days) until enough precursor cells have been collected.

Once precursor cells are isolated or collected, the blood can beprocessed to produce an enriched precursor cells population. Enrichedprecursor cells produced from umbilical CB or placental blood can form apopulation of CB stem cells. Preferably, the enriched precursor cellsare enriched in CD34+ HSC (and, thus, T cell depleted). Enrichment thuscan refer to a process wherein the percentage of HSC in the sample isincreased (relative to the percentage in the sample before theenrichment procedure). Purification is one example of enrichment. Incertain embodiments, the increase in the number of CD34+ cells (or othersuitable antigen-positive cells) as a percentage of cells in theenriched sample, relative to the sample prior to the enrichmentprocedure, is at least 25-, 50-, 75-, 100-, 150-, 200, 250-, 300-,350-fold, and preferably is 100-200 fold. In a preferred embodiment, theCD34+ cells are enriched using a monoclonal antibody to CD34, whichantibody is conjugated to a magnetic bead, and a magnetic cellseparation device to separate out the CD34+ cells. In some embodiments,using anti-CD34 antibodies, HSC are enriched from 1-2% of a normal bonemarrow cell population to approximately 50-80% of the population, asdescribed in sec. 5.4.1.1 of U.S. Pat. No. 7,399,633.

Any technique known in the art for cell separation/selection can be usedto carry out enrichment for a cell type such as HSC. For example,methods which rely on differential expression of cell surface markerscan be used.

Procedures for separation may include magnetic separation, usingantibody-coated magnetic beads; fluorescence activated cell sorting(FACS); affinity chromatography; cytotoxic agents joined to a monoclonalantibody or used in conjunction with a monoclonal antibody, e.g.,complement and cytotoxins; and “panning” with antibody attached to asolid matrix, e.g., plate, or other convenient technique. Techniquesproviding accurate separation/selection include fluorescence activatedcell sorters, which can have varying degrees of sophistication, e.g., aplurality of color channels, low angle and obtuse light scatteringdetecting channels, impedance channels, etc.

Antibodies may be conjugated with markers, such as magnetic beads, whichallow for direct separation, biotin, which can be removed with avidin orstreptavidin bound to a support, fluorochromes, which can be used with afluorescence activated cell sorter, or the like, to allow for ease ofseparation of the particular cell type. Any technique may be employedwhich is not unduly detrimental to the viability of the remaining cells.In one embodiment, the enrichment of HSC is affected by contacting aprecursor cell sample with a solid substrate (e.g., beads, flask,magnetic particles) to which antibodies are bound, and by removing anyunbound cells, wherein the HSC can be found either in the cells bound tothe solid substrate or in the unbound cells depending on the antibodiesused.

In one embodiment of the present disclosure, a precursor cell sample(e.g., a fresh CB unit) is processed to select for, i.e., enrich for,CD34+ cells using anti-CD34 antibodies directly or indirectly conjugatedto magnetic particles in connection with a magnetic cell separator, forexample, the CliniMACS® Cell Separation System (Miltenyi Biotec,Bergisch Gladbach, Germany), which employs nano-sized super-paramagneticparticles composed of iron oxide and dextran coupled to specificmonoclonal antibodies. The CliniMACS® Cell Separator is a closed sterilesystem, outfitted with a single-use disposable tubing set.

Similarly, CD133+ cells can be enriched using anti-CD133 antibodies. Ina specific embodiment, CD34+CD90+ cells are enriched for. Similarly,cells expressing CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD166,HLA DR, or a combination of the foregoing, can be enriched for usingantibodies against the antigen.

In one embodiment, HSC express CD34 (CD34+) and lack CD38 expression(CD38). In some embodiments, HSC are selected and/or enriched forCD34+CD38− cells. In specific embodiments, HSC are CD34+ and CD3T,CD38−, HLA DK and/or Thy-1^(lo). In some embodiments, HSC are selectedand/or enriched for CD34+ and CD33, CD38−, HLA DK and/or Thy-1^(lo)cells. In particular embodiments, human HSC are CD45Ra−, CD19− and/orc-kit+. In some embodiments, HSC are selected and/or enriched forCD45Ra−, CD19− and/or c-kit+ cells. In one embodiment, HSC expressvascular endothelial growth factor receptor 2 (VEGFR2). In someembodiments, HSC are selected and/or enriched for VEGFR2, which can beused as a marker for HSC.

HSC can also be enriched as described in sec. 5.4.1.1 of U.S. Pat. No.7,399,633. In particular, human HSC can be enriched by incubating asample with antibodies that recognize one or more of glycophorin A, CD3,CD24, CD16, CD14, CD34, CD45Ra, CD36, CD56, CD2, CD19, CD20, CD66a andCD66b, and separating the antibody-bound cells from non-antibody boundcells. In some of these embodiments, the non-antibody bound cellpopulation would be enriched for HSC. In some embodiments My10 andHLA-DR are used to obtain enriched HSC. In some embodiments, Tlymphocyte depletion is used to enrich for HSC, e.g., by pretreatingcells with a monoclonal antibody that recognizes a T cell antigen pluscomplement. In one embodiment, glycophorin A antibody is used to selectfor or against erythrocytes. In other embodiments, antibodies againstCD14, CD16, CD66a and CD66b are used to select for or against monocytes.In other embodiments, antibodies against CD24, CD3, CD19, CD20, CD56,CD2 are used to select for or against B and T lymphocytes and NK cells.In yet another embodiment, antibodies against CD45RA and CD36 are usedto select for or against T-cells, B-cells, granulocytes, platelets,monocytes, differentiated erythroid precursors, and some committedmature progenitors. Markers of pre-B progenitor cells can be MHC classII antigens. CD21 is a marker of mature B cells. In specificembodiments, antibodies which can be used for enrichment of HSC includeMy-10 and 3C5 (which recognize CD34), or RFB-1 (which recognizes CD99and identifies populations of BFU-E cells). Other antibodies against theabove-mentioned hematopoietic antigens are disclosed in U.S. Pat. No.5,877,299.

The above-mentioned antibodies can be used alone or in combination withprocedures such as “panning” (Broxmeyer et al., 1984, J. Clin. Invest.73:939-953) or fluorescence activated cell-sorting (FACS) (Williams etal., 1985, J. Immunol. 135:1004; Lu et al., 1986, Blood 68(1):126-133)to isolate the cells containing surface determinants recognized by theseantibodies, as described in sec. 5.4.1.1 of U.S. Pat. No. 7,399,633.

In a specific embodiment, the HSC (e.g., from umbilical CB and/orplacental blood) sample are red cell depleted, and the number of CD34+cells in the red cell depleted fraction is calculated. Preferably, theHSC (e.g. umbilical CB and/or placental blood) samples containing morethan 3.5 million CD34+ cells are enriched by the enrichment methodsdescribed above. After HSC have been isolated according to theenrichment methods described above or other methods known in the art,the enriched HSC can be expanded in order to increase the number of HSC,e.g., CD34+ cells. In less preferred embodiments, the methods describedherein can be applied without prior enrichment, or prior to enrichment.

In some embodiments, precursor cells that are subjected to expansionusing the methods described herein are fresh, i.e., they have not beenpreviously cryopreserved and thawed. In other embodiments, precursorcells that are subjected to expansion using the methods described hereinhave been cryopreserved and thawed. The precursor cells can be derived,e.g., from peripheral blood (such as mobilized peripheral blood), bonemarrow, umbilical CB, or placental blood.

1.7 Methods of Use

Methods disclosed herein include treating subjects (humans, veterinaryanimals (dogs, cats, reptiles, birds, etc.) livestock (horses, cattle,goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice,fish, etc.) with precursor cells expanded as disclosed herein. Treatingsubjects includes delivering therapeutically effective amounts.Therapeutically effective amounts include those that provide effectiveamounts, prophylactic treatments and/or therapeutic treatments.

An “effective amount” is the amount of precursor cells necessary toresult in a desired physiological change in the subject. Effectiveamounts are often administered for research purposes. In particularembodiments, an effective amount results in an increased number of SCIDrepopulating cells in an expanded sample determined by limiting-dilutionanalysis as shown by enhanced engraftment in NOD/SCID mice infused withthe expanded sample, relative to that seen with the unexpanded sample,where the unexpanded sample and expanded sample are from differentaliquots of the same sample, wherein the expanded sample but not theunexpanded sample is subjected to the expansion technique. Effectiveamounts can also be determined using animal models for long-termengrafting potential of HSC such as the SCID-hu bone model (Kyoizumi etal. (1992) Blood 79:1704; Murray et al. (1995) Blood 85(2) 368-378) andthe in utero sheep model (Zanjani et al. (1992) J. Clin. Invest.89:1179)). For a review of animal models of human hematopoiesis, seeSrour et al. (1992) J. Hematother. 1:143-153 and the references citedtherein. Effective amounts can also be assessed in vitro models such asthe long-term culture-initiating cell (LTCIC) assay, based on a limitingdilution analysis of the number of clonogenic cells produced in astromal co-culture after 5 to 8 weeks (Sutherland et al. (1990) Proc.Nat'l Acad. Sci. 87:3584-3588). The LTCIC assay has been shown tocorrelate with another commonly used stem cell assay, the cobblestonearea forming cell (CAFC) assay, and with long-term engrafting potentialin vivo (Breems et al. (1994) Leukemia 8:1095).

A “prophylactic treatment” includes a treatment administered to asubject who does not display signs or symptoms of a deficient cellpopulation or displays only early signs or symptoms of a deficient cellpopulation such that treatment is administered for the purpose ofdiminishing, preventing, or decreasing the risk of developing thedeficient cell population further. Thus, a prophylactic treatmentfunctions as a preventative treatment against a deficient cellpopulation.

A “therapeutic treatment” includes a treatment administered to a subjectwho displays symptoms or signs of a deficient cell population and isadministered to the subject for the purpose of diminishing oreliminating those signs or symptoms of the deficient cell population.The therapeutic treatment can reduce, control, or eliminate the presenceof the deficient cell population.

For administration, therapeutically effective amounts (also referred toherein as doses) can be initially estimated based on results from invitro assays and/or animal model studies. The actual dose amountadministered to a particular subject can be determined by a physician,veterinarian or researcher taking into account parameters such asphysical and physiological factors including target, body weight,severity of cell deficiency, previous or concurrent therapeuticinterventions, idiopathy of the subject and route of administration.

Therapeutically effective amounts can include as few as several hundredcells (or fewer) to as many as several million or more. In specificembodiments, therapeutically effective amounts range from 10³ to 10⁸cells per 100 kg. In additional embodiments, and in human subjects,therapeutically effective amounts can include between 10³ to 10⁸ cellsper 100 kg per infusion; 10⁴ to 10⁹ cells per 100 kg per infusion; or10⁵ and 10¹³ cells per 100 kg per infusion. In another embodiment,therapeutically effective amounts can include 1×10⁸ to 5×10¹² cells per100 kg per infusion. In another embodiment, therapeutically effectiveamounts can include between 1×10⁹ and 5×10¹¹ cells per 100 kg person perinfusion. For example, dosages such as 4×10⁹ cells per 100 kg and 2×10¹¹cells can be infused per 100 kg.

In some embodiments, a single administration of precursor cells areprovided. In other embodiments, multiple administrations are used.Multiple administrations can be provided over periodic time periods suchas an initial treatment regime of 3 to 7 consecutive days, and thenrepeated at other times.

Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,and epidural routes. The precursor cells may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Intravenousadministration also affords ease, convenience and comfort at higherlevels than other modes of administration. In certain applications,systemic administration by intravenous infusion is more effectiveoverall. In another embodiment, the precursor cells are administered toan individual by infusion into the superior mesenteric artery or celiacartery. The precursor cells may also be delivered locally by irrigationdown the recipient's airway or by direct injection into the mucosa ofthe intestine.

The expanded precursor cells can be used for a variety of applications.In one embodiment, the precursor cells are used for transplantation,sometimes referred to as cell-based therapies or cell replacementtherapies, such as bone marrow transplants, gene therapies, tissueengineering, and in vitro organogenesis. As one example, hematopoieticprogenitor cell expansion for bone marrow transplantation is a potentialapplication of human bone marrow cultures. Human autologous andallogeneic bone marrow transplantation are currently used as therapiesfor diseases such as leukemia, lymphoma, and other life-threateningdiseases. For these procedures, however, a large amount of donor bonemarrow must be removed to ensure that there are enough cells forengraftment. The methods of the present disclosure circumvent thisproblem. Methods of transplantation are known to those skilled in theart.

Several terms are used herein with respect to transplantation therapies,also known as cell-based therapies or cell replacement therapy. Theterms autologous transfer, autologous transplantation, autograft and thelike refer to treatments wherein the cell donor is also the recipient ofthe cell replacement therapy. The terms allogeneic transfer, allogeneictransplantation, allograft and the like refer to treatments wherein thecell donor is of the same species as the recipient of the cellreplacement therapy, but is not the same individual. A cell transfer inwhich the donor's cells have been histocompatibly matched with arecipient is sometimes referred to as a syngeneic transfer. The termsxenogeneic transfer, xenogeneic transplantation, xenograft and the likerefer to treatments wherein the cell donor is of a different speciesthan the recipient of the cell replacement therapy.

Transplantation of precursor cells may be useful in the treatment ofhematopoietic disorders and diseases. In one embodiment, the precursorcells are used to treat or prevent a hematopoietic disorder or diseasecharacterized by a failure or dysfunction of normal blood cellproduction and maturation cell. In another embodiment, the precursorcells are used to treat or prevent a hematopoietic disorder or diseaseresulting from a hematopoietic malignancy. In yet another embodiment,the precursor cells are used to treat or prevent a hematopoieticdisorder or disease resulting from immunosuppression, particularlyimmunosuppression in subjects with malignant, solid tumors. In yetanother embodiment, the precursor cells are used to treat or prevent anautoimmune disease affecting the hematopoietic system. In yet anotherembodiment, the precursor cells are used to treat or prevent a geneticor congenital hematopoietic disorder or disease.

Additional examples of particular hematopoietic diseases and disorderswhich can be treated by precursor cells expanded by the methodsdisclosed herein include: (i) diseases resulting from a failure ordysfunction of normal blood cell production and maturation (e.g.,hyperproliferative stem cell disorders; aplastic anemia; pancytopenia;agranulocytosis; thrombocytopenia; red cell aplasia; Blackfan-Diamondsyndrome due to drugs, radiation, or infection idiopathic); (ii)Hematopoietic malignancies (e.g., acute lymphoblastic (lymphocytic)leukemia; chronic lymphocytic leukemia; acute myelogenous leukemia;chronic myelogenous leukemia; acute malignant myelosclerosis; multiplemyeloma; polycythemia vera; agnogenic myelometaplasia; Waldenstrom'smacroglobulinemia; Hodgkin's lymphoma; non-Hodgkin's lymphoma); (iii)immunosuppression in patients with malignant, solid tumors (e.g.,malignant melanoma; carcinoma of the stomach; ovarian carcinoma; breastcarcinoma; small cell lung carcinoma; retinoblastoma; testicularcarcinoma; glioblastoma; rhabdomyosarcoma; neuroblastoma; Ewing'ssarcoma; lymphoma; (iv) autoimmune diseases (e.g., rheumatoid arthritis;diabetes type I; chronic hepatitis; multiple sclerosis; systemic lupuserythematosus; (v) genetic (congenital) disorders (e.g., anemias;familial aplastic; Fanconi's syndrome; Bloom's syndrome; pure red cellaplasia (PRCA); dyskeratosis congenital; Blackfan-Diamond syndrome;congenital dyserythropoietic syndromes I-IV; Chwachmann-Diamondsyndrome; dihydrofolate reductase deficiencies; formamino transferasedeficiency; Lesch-Nyhan syndrome; congenital spherocytosis; congenitalelliptocytosis; congenital stomatocytosis; congenital Rh null disease;paroxysmal nocturnal hemoglobinuria; G6PD (glucose-6-phosphatedehydrogenase) variants 1, 2, 3; pyruvate kinase deficiency; congenitalerythropoietin sensitivity deficiency; sickle cell disease and trait;thalassemia alpha, beta, gamma; met-hemoglobinemia; congenital disordersof immunity; severe combined immunodeficiency disease (SCID); barelymphocyte syndrome; ionophore-responsive combined immunodeficiency;combined immunodeficiency with a capping abnormality; nucleosidephosphorylase deficiency; granulocyte actin deficiency; infantileagranulocytosis; Gaucher's disease; adenosine deaminase deficiency;Kostmann's syndrome; reticular dysgenesis; congenital leukocytedysfunction syndromes); (vi) others (e.g., osteopetrosis;myelosclerosis; acquired hemolytic anemias; acquired immunodeficiencies;infectious disorders causing primary or secondary immunodeficienciesbacterial infections (e.g., Brucellosis, Listerosis, tubercu-losis,leprosy); parasitic infections (e.g., malaria, Leishmaniasis); fungalinfections; disorders involving disproportions in lymphoid cell sets andimpaired immune functions due to aging; phagocyte disorders; Kostmann'sagranulocytosis; chronic granulomatous disease; Chediak-Higachisyndrome; neutrophil actin deficiency; neutrophil membrane GP-180deficiency; metabolic storage diseases; mucopolysaccharidoses;mucolipidoses; miscellaneous disorders involving immune mechanisms;Wiskott-Aldrich Syndrome; al-antitrypsin deficiency).

Expanded precursor cells are also useful as a source of cells forspecific hematopoietic lineages. The maturation, proliferation anddifferentiation of expanded hematopoietic cells into one or moreselected lineages may be effected through culturing the cells withappropriate factors including, but not limited to, erythropoietin (EPO),colony stimulating factors, e.g., GM-CSF, G-CSF, or M-CSF, SCF,interleukins, e.g., IL-1, -2, -3, -4, -5, -6, -7, -8, -13, etc., or withstromal cells or other cells which secrete factors responsible for stemcell regeneration, commitment, and differentiation. As is understood byone of ordinary skill in the art, differentiated precursor cells may beused in accordance with the methods of use described herein.

The disclosure includes formulating expanded precursor cells foradministration to a subject as a pharmaceutical composition comprising atherapeutically effective amount of the precursor cells. In oneembodiment, the precursor cells are substantially purified. Formulationof pharmaceutical compositions is well known in the art. In particularembodiments, pharmaceutical compositions can include pharmaceuticallyacceptable carrier or excipients such as saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof.Pharmaceutical compositions can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. The pharmaceuticalcomposition can be a liquid solution, suspension, or emulsion. Inparticular embodiments, pharmaceutical compositions can also include asolubilizing agent and/or a local anesthetic such as lignocaine to easepain at a site of the injection.

2. EXEMPLARY EMBODIMENTS

1. A method for expanding precursor cells comprising culturing theprecursor cells in the presence of a media comprising an immobilizedLILRB2 agonist with a molecular weight of at least 225 kD wherein theculturing is effective to maintain expansion of the precursor cellsbeyond a time at which precursor cells cultured in the media lacking animmobilized LILRB2 agonist with a molecular weight of at least 225 kDstop proliferating and/or die.2. A method of embodiment 1 wherein the LILRB2 agonist has a molecularweight of at least 250 kD3. A method of embodiment 1 or 2 wherein the LILRB2 agonist ismultimerized.4. A method of embodiments 1, 2 or 3 wherein the LILRB2 agonist is anangiopoietin-like protein (Angptl) or fragment thereof.5. A method of embodiment 4 wherein the Angptl or fragment thereofcomprises the coiled-coil domain of the Angptl.6. A method of embodiment 4 or 5 wherein the LILRB2 agonist is Angptl 1or a fragment thereof, Angptl2 or a fragment thereof, Angptl3 or afragment thereof, Angptl4 or a fragment thereof, Angptl5 or a fragmentthereof, Angptl7 or a fragment thereof, or Mfap4 or a fragment thereof.7. A method of embodiment 1 wherein the LILRB2 agonist is an LILRB2receptor antibody.8. A method of embodiment 7 wherein the LILRB2 antibody is a monoclonalantibody or a polyclonal antibody.9. A method of any one of embodiments 1-8 wherein the LILRB2 agonistbinds the Ig1 domain of LILRB2.10. A method of any of embodiments 1-8 wherein the LILRB2 agonist bindsthe Ig4 domain of LILRB2.11. A method of any of embodiments 1-8 wherein the LILRB2 agonist bindsthe Ig1 domain and the Ig4 domain of LILRB2.12. A method of any of embodiments 1-8 wherein the LILRB2 agonist bindsthe amino acids at positions 92-100 of LILRB2 within the Ig1 domain.13. A method of any of embodiments 1-8 wherein the LILRB2 agonist bindsthe amino acids at positions 94, 95 and/or 96 of LILRB2 within the Ig1domain.14. A method of any of embodiments 1-8 wherein the LILRB2 agonist bindsthe amino acids at positions 390-396 of LILRB2 within the Ig4 domain.15. A method of any of embodiments 1-8 wherein the LILRB2 agonist bindsthe amino acids at 392 and 393 of LILRB2 within the Ig4 domain.16. A method of any of embodiments 1-15 wherein the media is serum free.17. A method of any of embodiments 1-16 wherein the media comprises1-100 μg/ml immobilized LILRB2 agonist.18. A method of embodiment 17 wherein the media comprises 25 μg/mlimmobilized LILRB2 agonist.19. A method of embodiment 17 or 18 wherein the media further comprisesSCF; TPO and Flt3-ligand.20. A method of embodiment 17 or 18 wherein the media further comprises0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100ng/ml SCF; 0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or 100 ng/ml TPO and 0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75,1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95 or 100 ng/ml Flt3-ligand.21. A method of embodiment 20 wherein the media comprises 10 ng/ml SCF.22. A method of embodiment 20 wherein the media comprises 5 ng/ml TPO.23. A method of embodiment 20 wherein the media comprises 10 ng/mlFlt3-ligand.24. A method of embodiment 20 wherein the media comprises growthfactors: 10 ng/ml SCF; 5 ng/ml TPO; and 10 ng/ml Flt3-ligand.25. A method of any one of embodiments 1-25 wherein the precursor cellsare hematopoietic stem cells or hematopoietic progenitor cells.26. A method of any one of embodiments 1-25 wherein the precursor cellsare hematopoietic stem cells.27. A method of any one of embodiments 1-25 wherein the precursor cellsare obtained from bone marrow, umbilical cord blood, placental blood, orWharton's jelly.28. A method of any one of embodiments 1-25 wherein the precursor cellsare obtained from fetal or neonatal blood.29. A method for expanding precursor cells comprising culturing theprecursor cells in the presence of a media comprising an LILRB2 agonistand a Notch agonist wherein the culturing is effective to maintainexpansion of the precursor cells beyond a time at which precursor cellscultured in the media lacking an LILRB2 agonist and a Notch agonist stopproliferating and/or die.30. A method of embodiment 29 wherein the LILRB2 agonist is immobilizedin the culture media.31. A method of embodiment 29 or 30 wherein the Notch agonist isimmobilized in the culture media.32. A method of embodiment 29, 30 or 31 wherein the LILRB2 agonist has amolecular weight of at least 225 kD or at least 250 kD.33. A method of any one of embodiments 29-32 wherein the LILRB2 agonistis multimerized.34. A method of any one of embodiments 29-33 wherein the LILRB2 agonistis an angiopoietin-like protein (Angptl) or fragment thereof.35. A method of embodiment 34 wherein the Angptl or fragment thereofcomprises the coiled-coil domain of the Angptl.36. A method of embodiment 34 or 35 wherein the LILRB2 agonist is Angptl1 or a fragment thereof, Angptl2 or a fragment thereof, Angptl3 or afragment thereof, Angptl4 or a fragment thereof, Angptl5 or a fragmentthereof, Angptl7 or a fragment thereof, or Mfap4 or a fragment thereof.37. A method of any one of embodiments 29-33 wherein the LILRB2 agonistis an LILRB2 receptor antibody.38. A method of embodiment 37 wherein the LILRB2 antibody is amonoclonal antibody or a polyclonal antibody.39. A method of any one of embodiments 29-38 wherein the LILRB2 agonistbinds the Ig1 domain of LILRB2.40. A method of any one of embodiments 29-38 wherein the LILRB2 agonistbinds the Ig4 domain of LILRB2.41. A method of any one of embodiments 29-38 wherein the LILRB2 agonistbinds the Ig1 domain and the Ig4 domain of LILRB2.42. A method of any one of embodiments 29-38 wherein the LILRB2 agonistbinds the amino acids at positions 92-100 of LILRB2 within the Ig1domain.43. A method of any one of embodiments 29-38 wherein the LILRB2 agonistbinds the amino acids at positions 94, 95 and/or 96 of LILRB2 within theIg1 domain.44. A method of any one of embodiments 29-38 wherein the LILRB2 agonistbinds the amino acids at positions 390-396 of LILRB2 within the Ig4domain.45. A method of any one of embodiments 29-38 wherein the LILRB2 agonistbinds the amino acids at 392 and 393 of LILRB2 within the Ig4 domain.46. A method of any one of embodiments 29-45 wherein the Notch agonistis an extracellular, Notch-interacting domain of a Delta protein.47. A method of any one of embodiments 29-45 wherein the Notch agonistis Delta^(ext-IgG).48. A method of any one of embodiments 29-45 wherein the Notch agonistis in dimeric form.49. A method of any one of embodiments 29-49 wherein the media is serumfree.50. A method of any one of embodiments 29-50 wherein the media comprises0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100μg/ml immobilized LILRB2 agonist.51. A method of embodiment 50 wherein the media comprises 25 μg/mlimmobilized LILRB2 agonist.52. A method of embodiment 50 wherein the media comprises 0.025, 0.050,0.075, 0.01, 0.05, 0.08 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μg/mlimmobilized LILRB2 agonist.53. A method of any one of embodiments 29-52 wherein the media comprises0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100μg/ml Notch agonist.54. A method of embodiment 53 wherein the media comprises 0.5 to 2.5μg/ml Notch agonist.55. A method of any one of embodiments 29-52 wherein the media furthercomprises SCF; TPO and Flt3-ligand.56. A method of embodiment 55 wherein the media further comprises 0.025,0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng/ml SCF;0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100ng/ml TPO and 0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3,4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 ng/ml Flt3-ligand.57. A method of embodiment 56 wherein the media comprises 10 ng/ml SCF.58. A method of embodiment 56 wherein the media comprises 5 ng/ml TPO.59. A method of embodiment 56 wherein the media comprises 10 ng/mlFlt3-ligand.60. A method of embodiment 56 wherein the media comprises 10 ng/ml SCF;5 ng/ml TPO; and 10 ng/ml Flt3-ligand.61. A method of any one of embodiments 29-52 wherein the media furthercomprises SCF, Flt3-ligand, IL-6, TPO, FGF1 and IL-3.62. A method of embodiment 61 wherein the media comprises SCF,Flt3-ligand, IL-6, TPO, FGF1, IL-3 and heparin.63. A method of embodiment 61 wherein the media further comprises 0.025,0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng/ml SCF,0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100ng/ml Flt3-ligand, 0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 100 ng/ml IL-6, 0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5,0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or 100 ng/ml TPO, 0.025, 0.050, 0.075, 0.01, 0.05,0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95 or 100 ng/ml FGF1 and 0.025, 0.050,0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng/ml IL-3.64. A method of embodiment 63 wherein the media further comprises 0.025,0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μg/mlheparin.65. A method of embodiment 61 wherein the media further comprises 50ng/ml SCF, 50 ng/ml Flt3-ligand, 50 ng/ml IL-6, 50 ng/ml TPO, 20 ng/mlFGF1 and 10 ng/ml IL-3.66. A method of embodiment 65 wherein the media further comprises 10μg/ml heparin.67. A method of any one of embodiments 29-66 wherein the media furthercomprises retronectin.68. A method of embodiment 67 wherein the media further comprises 0.025,0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μg/mlretronectin.69. A method of embodiment 68 wherein the media further comprises 5μg/ml retronectin.70. A method of any one of embodiments 29-69 wherein the precursor cellsare hematopoietic stem cells or hematopoietic progenitor cells.71. A method of any one of embodiments 29-69 wherein the precursor cellsare hematopoietic stem cells.72. A method of any one of embodiments 29-69 wherein the precursor cellsare obtained from bone marrow, umbilical cord blood, placental blood, orWharton's jelly.73. A method any one of embodiments 29-69 wherein the precursor cellsare obtained from fetal or neonatal blood.74. A method for producing precursor cells for hematopoietictransplantation comprising culturing the precursor cells in the presenceof a media comprising an immobilized LILRB2 agonist with a molecularweight of at least 225 kD wherein the culturing is effective to produceprecursor cells suitable able to treat a subject when formulated foradministration and administered in an effective amount.75. A method of embodiment 74 wherein the LILRB2 agonist has a molecularweight of at least 250 kD76. A method of embodiment 74 or 75 wherein the LILRB2 agonist ismultimerized.77. A method of embodiment 74, 75 or 76 wherein the LILRB2 agonist is anangiopoietin-like protein (Angptl) or fragment thereof.78. A method of embodiment 77 wherein the Angptl or fragment thereofcomprises the coiled-coil domain of the Angptl.79. A method of embodiment 77 or 78 wherein the LILRB2 agonist is Angptl1 or a fragment thereof, Angptl2 or a fragment thereof, Angptl3 or afragment thereof, Angptl4 or a fragment thereof, Angptl5 or a fragmentthereof, Angptl7 or a fragment thereof, or Mfap4 or a fragment thereof.80. A method of embodiment 74 wherein the LILRB2 agonist is an LILRB2receptor antibody.81. A method of embodiment 80 wherein the LILRB2 antibody is amonoclonal antibody or a polycloncal antibody.82. A method of any one of embodiments 74-81 wherein the LILRB2 agonistbinds the Ig1 domain of LILRB2.83. A method of any one of embodiments 74-81 wherein the LILRB2 agonistbinds the Ig4 domain of LILRB2.84. A method of any one of embodiments 74-81 wherein the LILRB2 agonistbinds the Ig1 domain and the Ig4 domain of LILRB2.85. A method of any one of embodiments 74-81 wherein the LILRB2 agonistbinds the amino acids at positions 92-100 of LILRB2 within the Ig1domain.86. A method of any one of embodiments 74-81 wherein the LILRB2 agonistbinds the amino acids at positions 94, 95 and/or 96 of LILRB2 within theIg1 domain.87. A method of any one of embodiments 74-81 wherein the LILRB2 agonistbinds the amino acids at positions 390-396 of LILRB2 within the Ig4domain.88. A method of any one of embodiments 74-81 wherein the LILRB2 agonistbinds the amino acids at 392 and 393 of LILRB2 within the Ig4 domain.89. A method of any one of embodiments 74-88 wherein the media is serumfree.90. A method of any one of embodiments 74-89 wherein the media comprises0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100μg/ml immobilized LILRB2 agonist.91. A method of embodiment 90 wherein the media comprises 25 μg/mlimmobilized LILRB2 agonist.92. A method of embodiment any one of embodiments 74-91 wherein themedia further comprises SCF; TPO and Flt3-ligand.93. A method of embodiment 92 wherein the media further comprises 0.025,0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng/ml SCF;0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100ng/ml TPO and 0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3,4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 ng/ml Flt3-ligand.94. A method of embodiment 93 wherein the media comprises 10 ng/ml SCF.95. A method of embodiment 93 wherein the media comprises 5 ng/ml TPO.96. A method of embodiment 93 wherein the media comprises 10 ng/mlFlt3-ligand.97. A method of embodiment 93 wherein the media comprises 10 ng/ml SCF;5 ng/ml TPO; and 10 ng/ml Flt3-ligand.98. A method of any one of embodiments 74-97 wherein the precursor cellsare hematopoietic stem cells or hematopoietic progenitor cells.99. A method of any one of embodiments 74-97 wherein the precursor cellsare hematopoietic stem cells.100. A method of any one of embodiments 74-97 wherein the precursorcells are obtained from bone marrow, umbilical cord blood, placentalblood, or Wharton's jelly.101. A method of any one of embodiments 74-97 wherein the precursorcells are obtained from fetal or neonatal blood.102. A method for expanding precursor cells comprising culturing theprecursor cells in the presence of a media comprising an LILRB2 agonistand a Notch agonist wherein the culturing is effective to maintainexpansion of the precursor cells beyond a time at which precursor cellscultured in the media lacking an LILRB2 agonist and a Notch agonist stopproliferating and/or die.103. A method of embodiment 102 wherein the LILRB2 agonist isimmobilized in the culture media.104. A method of embodiment 102 or 103 wherein the Notch agonist isimmobilized in the culture media.105. A method of embodiment 102, 103 or 104 wherein the LILRB2 agonisthas a molecular weight of at least 225 kD or at least 250 kD.106. A method of any one of embodiments 102-105 wherein the LILRB2agonist is multimerized.107. A method of any one of embodiments 102-106 wherein the LILRB2agonist is an angiopoietin-like protein (Angptl) or fragment thereof.108. A method of any one of embodiments 102-107 wherein the Angptl orfragment thereof comprises the coiled-coil domain of the Angptl.109. A method of any one of embodiments 102-108 wherein the LILRB2agonist is Angptl 1 or a fragment thereof, Angptl2 or a fragmentthereof, Angptl3 or a fragment thereof, Angptl4 or a fragment thereof,Angptl5 or a fragment thereof, Angptl7 or a fragment thereof, or Mfap4or a fragment thereof.110. A method of any one of embodiments 102-106 wherein the LILRB2agonist is an LILRB2 receptor antibody.111. A method of embodiment 110 wherein the LILRB2 antibody is amonoclonal antibody or a polycloncal antibody.112. A method of any one of embodiments 102-111 wherein the LILRB2agonist binds the Ig1 domain of LILRB2.113. A method of any one of embodiments 102-111 wherein the LILRB2agonist binds the Ig4 domain of LILRB2.114. A method of any one of embodiments 102-111 wherein the LILRB2agonist binds the Ig1 domain and the Ig4 domain of LILRB2.115. A method of any one of embodiments 102-111 wherein the LILRB2agonist binds the amino acids at positions 92-100 of LILRB2 within theIg1 domain.116. A method of any one of embodiments 102-111 wherein the LILRB2agonist binds the amino acids at positions 94, 95 and/or 96 of LILRB2within the Ig1 domain.117. A method of any one of embodiments 102-111 wherein the LILRB2agonist binds the amino acids at positions 390-396 of LILRB2 within theIg4 domain.118. A method of any one of embodiments 102-111 wherein the LILRB2agonist binds the amino acids at 392 and 393 of LILRB2 within the Ig4domain.119. A method of any one of embodiments 102-118 wherein the Notchagonist is an extracellular, Notch-interacting domain of a Deltaprotein.120. A method of any one of embodiments 102-118 wherein the Notchagonist is Delta^(ext-IgG).121. A method of any one of embodiments 102-118 wherein the Notchagonist is in dimeric form.122. A method of any one of embodiments 102-121 wherein the media isserum free.123. A method of any one of embodiments 102-122 wherein the mediacomprises 0.025-100 μg/ml immobilized LILRB2 agonist.124. A method of embodiment 123 wherein the media comprises 25 μg/mlimmobilized LILRB2 agonist.125. A method of embodiment any one of embodiments 102-124 wherein themedia comprises 0.08 to 25 μg/ml immobilized LILRB2 agonist.126. A method of any one of embodiments 102-125 wherein the mediacomprises 0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or 100 μg/ml Notch agonist.127. A method of embodiment 126 wherein the media comprises 0.5 to 2.5μg/ml Notch agonist.128. A method of any one of embodiments 102-127 wherein the mediafurther comprises SCF; TPO and Flt3-ligand.129. A method of embodiment 128 wherein the media further comprises0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100ng/ml SCF; 0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or 100 ng/ml TPO and 0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75,1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95 or 100 ng/ml Flt3-ligand.130. A method of embodiment 129 wherein the media comprises 10 ng/mlSCF.131. A method of embodiment 129 wherein the media comprises 5 ng/ml TPO.132. A method of embodiment 129 wherein the media comprises 10 ng/mlFlt3-ligand.133. A method of embodiment 129 wherein the media comprises: 10 ng/mlSCF; 5 ng/ml TPO; and 10 ng/ml Flt3-ligand.134. A method of any one of embodiments 102-127 wherein the mediafurther comprises SCF, Flt3-ligand, IL-6, TPO, FGF1 and IL-3.135. A method of embodiment 134 wherein the media comprises SCF,Flt3-ligand, IL-6, TPO, FGF1, IL-3 and heparin.136. A method of any one of embodiments 102-127 wherein the mediafurther comprises 0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 ng/ml SCF, 0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5,0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or 100 ng/ml Flt3-ligand, 0.025, 0.050, 0.075, 0.01,0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng/ml IL-6, 0.025, 0.050,0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng/ml TPO,0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100ng/ml FGF1 and 1-100 ng/ml IL-3.137. A method of embodiment 136 wherein the media further comprises0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100μg/ml heparin.138. A method of embodiment 136 or 137 wherein the media furthercomprises 50 ng/ml SCF, 50 ng/ml Flt3-ligand, 50 ng/ml IL-6, 50 ng/mlTPO, 20 ng/ml FGF1 and 10 ng/ml IL-3.139. A method of embodiment 138 wherein the media further comprises 10μg/ml heparin.140. A method of any one of embodiments 102-139 wherein the mediafurther comprises retronectin.141. A method of embodiment 140 wherein the media further comprises0.025, 0.050, 0.075, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100μg/ml retronectin.142. A method of embodiment 141 wherein the media further comprises 5μg/ml retronectin.143. A method of any one of embodiments 102-142 wherein the precursorcells are hematopoietic stem cells or hematopoietic progenitor cells.144. A method of any one of embodiments 102-142 wherein the precursorcells are hematopoietic stem cells.145. A method of any one of embodiments 102-142 wherein the precursorcells are obtained from bone marrow, umbilical cord blood, placentalblood, or Wharton's jelly.146. A method of any one of embodiments 102-142 wherein the precursorcells are obtained from fetal or neonatal blood.147. A chimeric receptor reporter system comprising cells expressing afusion protein comprising at least one extracellular domain of LILRB2and transmembrane and cytoplasmic domains of PILRβ wherein binding of anactivating ligand to the extracellular domain of LILRB2 results inreporter gene expression.148. A chimeric receptor reporter system of embodiment 147 wherein theat least one extracellular domain of LILRB2 is the Ig1 domain, the Ig2domain, the Ig3 domain and/or the Ig4 domain.149. A chimeric receptor reporter system of embodiment 147 wherein theat least one extracellular domain of LILRB2 is the Ig1 domain.150. A chimeric receptor reporter system of embodiment 147 wherein theIg1 domain includes amino acids 24-119 of LILB2.151. A chimeric receptor reporter system of embodiment 147 wherein theat least one extracellular domain of LILRB2 is the Ig2 domain.152. A chimeric receptor reporter system of embodiment 147 wherein theIg2 domain includes amino acids 120-219 of LILB2.153. A chimeric receptor reporter system of embodiment 147 wherein theat least one extracellular domain of LILRB2 is the Ig3 domain.154. A chimeric receptor reporter system of embodiment 147 wherein theIg3 domain includes amino acids 221-320 of LILB2.155. A chimeric receptor reporter system of embodiment 147 wherein theat least one extracellular domain of LILRB2 is the Ig4 domain.156. A chimeric receptor reporter system of embodiment 147 wherein theIg4 domain includes amino acids 321-458 of LILB2.157. A chimeric receptor reporter system of embodiment 147 wherein theat least one extracellular domain of LILRB2 includes the Ig1 domain andthe Ig2 domain.158. A chimeric receptor reporter system of embodiment 147 wherein theIg1 domain and the Ig2 domain includes amino acids 24-219 of LILB2.159. A chimeric receptor reporter system of embodiment 147 wherein theat least one extracellular domain of LILRB2 includes the Ig3 domain andthe Ig4 domain.160. A chimeric receptor reporter system of embodiment 147 wherein theIg3 domain and the Ig4 domain includes amino acids 221-458 of LILB2.161. A chimeric receptor reporter system of embodiment 147 wherein thefusion protein includes amino acids 24-458 of LILRB2.162. A chimeric receptor reporter system of any one of embodiments147-161 wherein the cells are T cell hybridoma cells.163. A chimeric receptor reporter system of embodiment 162 whereinreporter gene expression results from NFAT activation.164. A chimeric receptor reporter system of embodiment 162 or 163wherein reporter gene expression is under the control of aNFAT-responsive promoter.165. A method of any one of embodiments 1-25 wherein the precursor cellsare embryonic stem cells; hematopoeitic precursors of HSC derived fromembryonic stem cells; induced pluripotent stem cells; or HSC orhematopoietic precursors derived by reprogramming.166. A method of any one of embodiments 29-69 wherein the precursorcells are embryonic stem cells; hematopoeitic precursors of HSC derivedfrom embryonic stem cells; induced pluripotent stem cells; or HSC orhematopoietic precursors derived by reprogramming.167. A method of any one of embodiments 74-97 wherein the precursorcells are embryonic stem cells; hematopoeitic precursors of HSC derivedfrom embryonic stem cells; induced pluripotent stem cells; or HSC orhematopoietic precursors derived by reprogramming.168. A method of any one of embodiments 102-142 wherein the precursorcells are embryonic stem cells; hematopoeitic precursors of HSC derivedfrom embryonic stem cells; induced pluripotent stem cells; or HSC orhematopoietic precursors derived by reprogramming.

The Examples below are included to demonstrate particular embodiments ofthe disclosure. Those of ordinary skill in the art should recognize inlight of the present disclosure that many changes can be made to thespecific embodiments disclosed herein and still obtain a like or similarresult without departing from the spirit and scope of the disclosure.

3. EXAMPLES 3.1 Example 1

A better understanding of the interaction between extrinsic factors andsurface receptors on HSC will greatly benefit stem cell research andapplications. Recently it was shown that several Angptls bind andactivate the immune inhibitory receptor LILRB2 to support ex vivoexpansion of HSC. However, the molecular basis for the interactionbetween Angptls and LILRB2 was unclear. Example 1 demonstrates thatAngptl2 expressed in mammalian cells forms high molecular weight (HMW)species, and ligand multimerization is required for activation of LILRB2for downstream signaling. A novel motif in the first and fourth Igdomains of LILRB2 was identified that is necessary for the receptor tobe bound and activated by Angptl2. The binding of Angptl2 to LILRB2 ismore potent than and not completely overlapped with the binding ofanother ligand HLA-G. Immobilized anti-LILRB2 antibodies induce a morepotent activation of LILRB2 than Angptl2. Example 1 describes aserum-free culture containing defined cytokines and immobilizedanti-LILRB2 that supports a net expansion of repopulating human CB HSCs.The elucidation of the mode of Angptl binding to LILRB2 enabled thedevelopment of this new approach for ex vivo expansion of human HSCs.

3.1.1. Example 1—Methods

Chimeric receptor reporter cells. The chimeric receptors includingindividual or all Ig-domains or their mutants of the extracellulardomain of LILRB2 and the transmembrane and cytoplasmic domains ofactivating PILRβ were infected into mouse T cell hybridoma cellscarrying NFAT-GFP reporter gene and DAP12 by using a retrovirus vector.Amino acid 24-458 of hLILRB2 was used to construct the full-lengthLILRB2 chimeric reporter. The amino acid sequences for the individual Igdomains or Ig combinations are: Ig1 (aa 24-119), Ig2 (aa 120-219), Ig3(aa 221-320), Ig4 (aa 321-458), Ig1+2 (aa 24-219), and Ig3+4 (aa221-458). These chimeric LILRB2-PILRβ receptor reporter cells(5×10⁴/well) were incubated with ligands for indicated time, and GFP wasanalyzed by flow cytometry. Purified Angptl2-FLAG by M2 resin or Angptl2in conditioned medium collected from transfected 293T cells were used asindicated. For experiments using coated wells, indicatedbacterially-expressed GST-Angptl2, anti-LILRB2 polyclonal antibody (pAb,#BAF2078, R&D systems), or anti-LILRB2 monoclonal antibody (mAb,#16-5149-85, eBioscience) or control antibody was pre-coated on 96well-plates for 3 hrs in 37° C. unless otherwise indicated. Whenantibodies were cross-linked, 10 μg/ml pAb was incubated with 10 μg/mlstreptavidin at 4° C. overnight.

Mice. NOD/SCID IL2R gamma−/−(NSG) mice were purchased and maintained atthe University of Texas Southwestern Medical Center animal facilities.All animal experiments were performed with the approval of UTSouthwestern Committee on Animal Care.

Plasmids and proteins. Wild-type or mutant LILRB2s were constructed intopLVX-IRES-ZsGreen (Clontech). Plasmid CMV-Kozak-human Angptl2 encodingAngptl2 or HLA-G extracellular domain (ECD) with a FLAG tag at theC-terminus was transfected into 293T cells using Lipofectamine 2000, andthe conditioned medium at 48 h was collected. Angptl2-FLAG was purifiedusing M2 resin. Bacterially-expressed GST-Angptls-FLAG was constructedin pGEX vector, and expressed and purified from bacteria. Concentrationsof Angptl proteins were adjusted to the same level for flowcytometry-based binding experiments. Purified recombinant HLA-G waspurchased from Origene (#TP305216).

SDS-PAGE and native polyacrylamide gel electrophoresis (PAGE). Forreduced SDS-PAGE, samples were mixed with 4×loading buffer withpmercaptoethanol (BME) and loaded on 10% SDS gels. For non-reducedSDS-PAGE, samples were mixed with 4×loading buffer without BME andloaded on 10% SDS gels. For native PAGE, the PAGE gel did not containSDS. Samples were mixed with 4×loading buffer without BME.

Fast protein liquid chromatography (FPLC). Purified GST-Angptl2expressed from bacteria was loaded onto a 16/60 Superdex 200 gelfiltration column and eluted with PBS and 2 mM EDTA. Fractions (0.6 ml)were collected, and the amount of Angptl2 in each fraction was analyzedby western blot analysis.

Human cell culture. Fresh and cryopreserved human CB cells werepurchased from AllCells. All of the cells were from pooled donors.Purities of CD34+ or CD133+ cells as analyzed by flow cytometry werehigher than 90%. After thawing, the cell viability tested by trypan blueexclusion was higher than 72%. The thawed cells were centrifuged andresuspended in StemSpan medium before being aliquoted for immediatetransplantation or culture. StemSpan supplemented with 50 ng/ml humanSCF, 10 ng/ml human TPO, and 50 ng/ml human Flt3-L with soluble orimmobilized anti-LILRB2 monoclonal antibody (#165149-85, eBioscience) orpolyclonal antibody (#BAF2078, R&D Systems) was used as culture medium.Fresh human CB CD34+ cells or cryopreserved CD133+ cells as indicatedwere plated at 5×10³ cells/well in one well of a U-bottom 96-well plate(3799; Corning) with 200 μl of the indicated medium for 2 days. On day3, cells were pooled from individual wells and transferred to 6-wellplates at 5×10⁴ cells/ml. Fresh medium was added at days 4 and 7 to keepthe cell density at 2×10⁵ cells/ml (day 4) or 7×10⁵ cells/ml (day 7).Cells were cultured at 37° C. in 5% CO₂ and normal O₂ or 5% O2 (low O₂)levels. For transplantation, cells from all the culture wells werepooled before the indicated numbers of cells were transplanted into eachmouse.

NSG transplant. Uncultured or cultured progenies of human CB CD133+ orCD34+ cells at indicated days were pooled together and the indicatedportions were injected intravenously via the retro-orbital route intosub-lethally irradiated (250 rad) 8-10 week old NSG mice. Eight weeks oras indicated after transplantation, bone marrow nucleated cells fromtransplanted animals were analyzed by flow cytometry for the presence ofhuman cells. For secondary transplantations, bone marrow aspirates fromone hind leg of a primary recipient were used to transplant twosecondary recipients as described in Bryder et al., Am J Pathol. 2006;169:338-346. Calculation of long-term repopulating cells (competitiverepopulating unit, CRU) in limiting dilution experiments was conductedusing L-Calc software (StemCell Technologies). See, for example,Brunstein and Wagner, Annu Rev Med. 2006; 57:403-417; Chou et al., CellStem Cell. 2010; 7:427-428; and Delaney et al., Nat Med. 2010;16:232-236.

For limiting dilution analysis, mice were considered to be positive forhuman HSC engraftment when at least 1% (for primary transplantation) or0.1% (for secondary transplantation) CD45/71+ human cells were detectedamong the mouse bone marrow cells, unless otherwise indicated.

Flow cytometry. To measure Angptl2/LILRB2 binding, plasmids forexpression of LILRB2 or mutants driven from a CMV promoter weretransfected into 293T cells. Cells were harvested at 48 h for analysis.Alternatively, mononuclear human CB cells were incubated with Fc blockand equal amounts of FLAG-tagged Angptl2 at 4° C. for 60 min, followedby staining with anti-Flag-APC and propidium iodide. Anti-LILRB2-PE wasused as indicated. Cells were analyzed using either a FACSCalibur orFACSAria instrument (Becton Dickinson).

Human hematopoietic engraftment in NSG mice was assessed following theprotocol described in Himburg et al., Nat Med. 2010; 16:475-482.Briefly, bone marrow cells from recipient NSG mice were stained withantihuman CD45-PE, CD71-PE, CD15-FITC, and CD66b-FITC to quantify thetotal human hematopoietic (CD45/71+) cell population as well as thesubset of exclusively granulopoietic (CD15/66b+) cells within thispopulation. Cells were stained with antihuman CD34-FITC and anti-humanCD19-PE and CD20-PE to quantify human progenitor (CD34+) and B-lineage(CD34−CD19/20+) populations. Anti-human CD3 was used to analyze humanT-lineage reconstitution. Anti-human CD34-FITC and anti-human CD90-APCwas used to quantitate CD34+ or CD34+CD90+ cells in culture. Allantihuman antibodies were purchased from Becton Dickinson.

Retrovirus infection. The retroviral plasmids with PCL-ECO (2:1) weretransfected using Lipofectamine 2000 (Invitrogen) into 293T cells. Theresulting retroviral supernatant was collected 48-72 hours later and wasused for infection. Target cells were resuspended in viral supernatants(1×10⁵ cells/ml) with 4 μg/ml polybrene and centrifuged at 2000 rpm for120 min before culturing for 24 hours in RPMI-1640 medium (Sigma) plus10% FBS and 100 U/ml penicillin/streptomycin. Cells were thenresuspended in viral supernatant for another round of infection.

Live cell immunofluorescence and confocal microscopy. The LILRB2chimeric receptor reporter cells were treated with PBS or coatedmonoclonal anti-LILRB2 antibody for 6 hours at 37° C. These cells werewashed by PBS twice and stained with rat anti-human LILRB2 antibody for15 minutes at 4° C. The cells were further stained with goat anti-ratCy3 for 15 minutes at 4° C. and mounted on a slide for examination by aZeiss LSM 710 confocal microscope. Each image was scanned in Z-seriesfrom top to bottom of cells.

3.1.2. Example 1—Results

Multimerized Angptl2 activates LILRB2. Because there is no definitivedownstream reporter for LILRB-mediated signaling, a stable reporter cellsystem was employed to test whether Angptl2 can bind to and activateLILRB2. In this chimeric receptor reporter system, the extracellulardomain (ECD) of LILRB2 is fused with transmembrane/intracellular domainsof paired immunoglobulin-like receptor β (PILRβ) that associates withthe adaptor protein DAP12 containing ITAM. When the chimeric receptor isactivated by Angptl2 binding to the ECD of LILRB2, ZAP70 or Syk kinaseis recruited to the ITAM of the adaptor DAP12 and activates the nuclearfactor of activated T cells (NFAT) to promote GFP expression driven bythe NFAT responsive promoter (FIG. 1A). The establishment of thisreporter was inspired by a LILRB1 reporter system (Arase et al.,Science. 2002; 296:1323-1326; Ohtsuka et al., Proc Natl Acad Sci USA.2004; 101:8126-8131) and it serves as a surrogate and sensitive systemto enable study of the signaling-induction abilities of different formsof Angptl2 (including soluble, immobilized, monomeric, and oligomeric)and to screen additional agonists and antagonists.

To test whether Angptl2 expressed in mammalian cells can activatesignaling through LILRB2, LILRB2 reporter cells were incubated withconditioned medium collected from 293T cells transfected with a plasmiddesigned to express Angptl2 (2 μg/ml) (schematic in FIG. 2A).Conditioned medium from mock transfected 293T cells served as thecontrol. After 24 h, Angptl2-treated LILRB2 reporter cells induced asignificantly greater GFP expression than the control cells (18.95±0.95%versus 5.34±1.19%; FIG. 1B). The potential binding/activation of LILRB2by the immobilized HLA-G was also measured using the same LILRB2reporter cells. GFP activation was not detected by as much as 130 μg/mlHLA-G (FIG. 3). This suggests that Angptl2 is capable of binding andactivating LILRB2, with a significantly greater ability than HLA-G. Inparallel, the binding of Angptl2/LILRB2 to HLA-G-LILRB2 using flowcytometry analysis. To this end, a secretable HLA-G-ECD expressionvector was constructed with the same signal peptide as Angptl2expression vector (FIG. 2A) and collected the same amount of solubleHLA-G-ECD and Angptl2 for binding to LILRB2 expressed on the surface oftransiently transfected 293T cells. Similar to the result of thechimeric reporter system, Angptl2 binds to LILRB2 with a significantlygreater affinity than HLAG (FIG. 1C).

In the described characterization of Angptl2 expressed in 293T cells,full-length (FL), coiled-coil domain (CC), and FBN-like domain ofAngptl2 with or without β-mecaptoethanol (BME) treatment were compared.BME treatment reduces disulfide bonds that stabilize a HMW form ofAngptl2. When analyzed by SDS-PAGE, full-length and CC domainpreparations of Angptl2 ran partially or exclusively, respectively, as aHMW band (larger than 250 kD), whereas the FBN domain migrated as a 37kD band, corresponding to the expected size of the FBN monomer (FIG.1D). The HMW species was likely in a multimerization state. Similarly,multimerized Angptl2 exists in mouse serum and plasma (FIG. 4).

A bacterial expression system enabled production and purification of alarge amount of GST-tagged Angptl2 (Zheng et al. Nature. 2012;485:656-660). The monomer GST-Angptl2 was purified by size exclusionchromatography and the product was detected in native-PAGE and westernblotting (FIGS. 1E-1G). Because the multimerized ligand can induce theclustering of surface receptor (Arase et al., Science. 2002;296:1323-1326; Ohtsuka et al., Proc Natl Acad Sci USA. 2004;101:8126-8131) and immobilized ligand may also cluster the receptor, theabilities of the soluble and immobilized monomeric GST-Angptl2 toactivate the LILRB2 reporter cells were compared. The monomericGST-Angptl2 was immobilized on the wells of the tissue culture plate oradded into the medium, and LILRB2 reporter cells were added. Only theimmobilized monomeric form, and not the soluble monomer, induced GFPexpression (20±2.65% versus 3.56±0.98%; FIG. 1H). Because the nativelymultimerized form of Angptl2 and the immobilized monomer Angptl2activated LILRB2 but the soluble monomeric Angptl2 did not, it wasconcluded that Angptl2 must be multimerized to become an active ligandof LILRB2.

To further study whether a multimerized form of Angptl2 is needed toactivate LILRB2, the effects of monoclonal and polyclonal anti-LILRB2antibodies on the LILRB2 reporter cells were tested. Both solublemonoclonal and polyclonal anti-LILRB2 blocked the activation of LILRB2by the immobilized Angptl2 (from 19.9% to 0.81 or 2.31%; FIG. 5A),supporting the idea that the binding between Angptl2 and LILRB2 is notas potent as that between anti-LILRB2 and LILRB2. Whereas neither thecontrol antibody nor the soluble anti-LILRB2 stimulated GFP expression,both immobilized monoclonal and polyclonal anti-LILRB2 efficientlyinduced upregulation of GFP (from 3.88% to 33.7 or 87%; FIG. 5B).Moreover, cross-linking of biotin-conjugated anti-LILRB2 bystreptavidin-activated GFP expression (from 3.1% to 36.4%; FIG. 5C).Although anti-LILRB2 has a higher binding affinity for LILRB2 than doesAngptl2, only immobilized but not soluble antibodies activated LILRB2.To determine if immobilized ligands induce receptor clustering, theLILRB2 localization on the cell surface with or without immobilizedmonoclonal anti-LILRB2 treatment was examined. Without treatment, themajority of LILRB2 chimeric reporter cells (81.2%) exhibited an evendistribution of LILRB2-ECD on the cell membrane, with a “Ring”-likeshape under confocal microscopy observation (FIG. 5D). By contrast,after treatment with immobilized antibodies, the distribution ofLILRB2-ECD was changed from the “Ring”-to the “Spot”-like shape in 97.8%signaling activated cells (indicated as GFP induced cells) (FIG. 5D).These results further support the conclusion that multimerized ligandsinduce the clustering of the receptor LILRB2 for signaling activation.

A motif in Ig domains of LILRB2 is critical for the effects of Angptl2in binding and receptor activation. LILRB2 contains four extracellularimmunoglobulin (Ig)-like domains. Takai et al., J Biomed Biotechnol.2011; 2011:275302. It was hypothesized that one or more of theseIg-domains bind to Angptl2. To test this hypothesis, a number of domainand site-specific mutations of LILRB2 were generated for the flowcytometry-based cell surface ligand binding assay and chimeric reporterassay. To start with, Angptl2 binding abilities of a number of domainmutations of LILRB2 were screened. Although individual Ig domains ofLILRB2 do not bind to Angptl2, Ig1 and 2 in combination, and Ig3 and Ig4in combination displayed about 50% and 10%, respectively, of the maximalbinding between the full-length LILRB2 and Angptl2 (FIGS. 6A-6B, FIG.7). This suggests that the major Angptl2 binding site resides in Ig1 andIg2 of LILRB2 and that Ig3 and Ig4 facilitate binding of C-terminaldomains of the protein.

Next a series of site-specific mutations in amino acids potentiallycritical to the binding of ligand to LILRB2 based on the structure ofLILRB2 28 (PDB structure 2GY7) were designed. Based on the physicalmapping data of LILRB2, both Ig1-Ig2 and Ig3-Ig4 combination canmaintain its binding activity with Angptl2, which indicates that thesingle interface on each Ig domain is not essential for LILRB2 bindingaffinity. For this type of Ig structure, the binding interface ispossibly located at flexible and variable loops rather than rigid andconserved beta-sheets. Based on the PDB structure of Ig1-Ig2 domain(PDBID: 2GW5 and 2DYP) and Ig3-Ig4 domain (PDBID: 4LLA), the possibleinterface on each Ig domain was designed (FIGS. 8A-8C). Because theoverall geometry of four Ig domains is highly flexible and each one ofit is not essential, the point mutation on each Ig domain may not blockthe overall binding between LILRB2 and Angptl2. Therefore, someadditional large and hydrophobic residues were identified formutagenesis study (FIGS. 8A-8B). However, these mutant LILRB2 do notsignificantly decrease Angptl2 binding (FIG. 8C). These results suggestthat Angptl2 may have somewhat different binding pockets in LILRB2 thanHLA-G (see below for additional data).

The bioinformatical analysis and mutagenesis study results were combinedto find that AA 92-100 in Ig1 is critical for Angptl2 binding. The Ig1+2fragment with mutations in this region (Mut-8 aa, SEQ ID NO: 20) did notbind to Angptl2, and Angptl2 binding of the full-length LILRB2 with thesame mutation was decreased by more than 50% relative to the wild-typeprotein (FIGS. 6C-6D). Further experiments showed that single mutationsin G94, R95, or Y96 each decreased the Angptl2 binding of full-lengthLILRB2 by half (FIG. 6E), indicating these three amino acids areessential for Angptl2 binding in Ig1. A similar motif, AA 390-396,exists in Ig4 (FIG. 6C, SEQ ID NO: 19). A single mutation in Y394decreased Angptl2 binding of full-length LILRB2 by about 30% (FIG. 6E).Furthermore, when combined with Y96A, G392D or Y394A mutations totallyabrogated the Angptl2 binding of full-length LILRB2 (FIGS. 6E-6F).Therefore, these results indicate that Ig2 helps Ig1 to form the majorAngptl2 binding site, Ig4 further facilitates Ig1+2 binding, and theH*G*Y*C motifs in Ig1 (SEQ ID NO: 18) and Ig4 (SEQ ID NO: 19) arecritical for LILRB2 to bind Angptl2.

To further investigate whether Angptl2, Angptl5, and HLA-G bind to thesame regions in LILRB2, the bindings of these ligands to mutant LILRB2were compared. HLA-G binds to a number of mutant LILRB2 including H92S,T93A, G94D, R95E, Q99R, G392D, T393E, and HLA-G binding site 1 (MHC-S1)(from the structures by Shiroishi et al., Proc Natl Acad Sci USA. 2006;103:16412-16417; Shiroishi et al., Proc Natl Acad Sci USA. 2003;100:8856-8861) with lower affinity than Angptl2. Angptl5 binds to mutantLILRB2 generally more similar to Angptl2 than to HLA-G (FIG. 6G, FIG.2B). Together with the result in FIG. 8C, the data suggest that thebinding of Angptl2 or HLA-G to LILRB2 is partially but not completelyoverlapped.

In addition to the flow cytometry-based binding analysis, the activationof various LILRB2 mutants treated with immobilized antibodies or Angptl2in the chimeric reporter system was measured. It was found that only Ig1and Ig2 domains in combination could bind and be activated by ligands(FIG. 6H). Moreover, a single mutation in Y96 led to a dramatic decreaseof GFP induction, and the combined mutations of Y96A with either G392Dor Y394A totally abrogated the GFP induction by immobilized Angptl2(FIG. 6I). Therefore the results in using chimeric reporter systemconfirmed that the H*G*Y*C motifs in Ig1 (SEQ ID NO: 18) and Ig4 (SEQ IDNO: 19) are essential for LILRB2 to bind Angptl2. Furthermore, theysuggest that the indicated motifs are critical for LILRB2 activation.

To identify the sites in Angptls that bind to LILRB2, the binding offull-length with CC and FBN domains of Angptl2 to LILRB2 was compared.The full-length protein, but not the CC or the FBN domain of Angptl2,bound to 293T cells that expressed LILRB2 21. The full-length proteinand the CC domain (but not FBN domain) of Angptl2 bound to LILRB2+ humanCB mononuclear cells (FIG. 9). The full-length Angptl2, the FBN domain,and a high concentration of soluble CC domain were able to activateLILRB2 reporter cells (FIG. 10). It is speculated that the actualconcentration of the CC domain coated on the plastic dish might be lowerthan that of the soluble CC domain, and therefore this immobilized CCdomain was not sufficiently high to activate the LILRB2 chimericreporter. These results suggest that both CC and FBN of Angptl2 areneeded for optimal binding and activation of LILRB2.

Anti-LILRB2 antibodies support ex vivo expansion of human CB HSCs.Although numerous conditions have been used for expansion of HSCs inculture, the optimal mixture of growth factors and cytokines to allowexpansion sufficient for clinically applicability has not yet beendetermined. See, for example, Chou et al., Cell Stem Cell. 2010;7:427-428; Delaney et al., Nat Med. 2010; 16:232-236; Himburg et al.,Nat Med. 2010; 16:475-482; Zheng et al., Cell Stem Cell. 2011;9:119-130; Boitano et al., Science. 2010; 329:1345-1348; Butler et al.,Cell Stem Cell. 2010; 6:251-264; North et al., Nature. 2007;447:1007-1011; Antonchuk et al., Cell. 2002; 109:39-45; Dahlberg et al.,2011; 117:6083-6090; Kirouac et al., Curr Opin Biotechnol. 2006;17:538-547; and Robinson et al., Cytotherapy. 2005; 7:243-250.

Previously Angptls were identified as growth factors for HSC expansion.Zheng et al., Blood. 2011; 117:470-479; Zhang et al., Blood. 2008;111:3415-3423 and Zhang et al., Nat Med. 2006; 12:240-245. However,because Angptls are large glycosylated proteins that are readilydegraded and form aggregates, they are difficult to express and purifyand thus are not ideal components for use in culture. Molecules withenhanced stability and higher activities that mimic the effects of theAngptls would lead to the development of a more efficient HSC expansionsystem. Based on the finding that immobilized antibody to LILRB2mimicked Angptl2-stimulated receptor signaling (FIGS. 5A-5D), whetherimmobilized anti-LILRB2 antibody would support ex vivo expansion ofhuman CB HSCs was tested.

Culture of human CB CD133+ cells in STF medium, medium with solubleanti-LILRB2, and medium with immobilized either polyclonal (pAb) ormonoclonal (mAb) anti-LILRB2 antibody was first compared. 1×10⁴cryopreserved human CB CD133+ cells were plated in indicated medium;after 10 days of culture the total numbers of cells were determined.More expansion resulted from culture with immobilized pAb or mAb thaneither in STF medium or STF medium containing soluble antibodies (230%and 125% of the STF sample value, respectively; FIGS. 11A-11B). It is ofnote that the levels of CD34+CD90+ cells that may be enriched incultured HSCs were similar in these conditions (FIG. 11C).

Colony-forming assays were next performed. Concordant with the cellgrowth results, samples of cells treated with the immobilized antibodieshad significantly increased colony forming units (CFUs) in both primaryand secondary colony forming assays than the STF sample or the solubleantibody sample (FIGS. 11D-11E). In particular, the immobilizedpolyclonal and monoclonal antibody treated samples had more than 3-foldand 1.6-fold of CFUs, respectively, than the STF samples in thesecondary replating.

Finally, cells cultured in these same conditions were transplanted intosublethally irradiated NSG mice (1×10⁴ input equivalent cells permouse). CD133+ cells cultured with immobilized anti-LILRB2 pAb had asignificantly greater average chimerism in peripheral blood and bonemarrow than the counterparts without antibody treatment or than cellstreated with soluble antibody at the analyzed post-transplant timepoints (3-36 weeks, FIGS. 12A-12B). FIG. 12C shows human hematopoieticengraftment at 36 weeks in representative mice that were transplantedwith differently cultured human CB CD133+ cells. Mice that weretransplanted with cells cultured in immobilized antibody displayed amuch higher engraftment of total hematopoietic (CD45/71+) (52.48±5.41%versus 23.13±7.93%; FIG. 12B), myeloid (CD15/66b+) (3.29±0.41% versus1.16±0.38%; FIG. 12D), B-lymphoid (CD34−CD19/20+) (27.43±5.15% versus8.52±2.58%; FIG. 12E), and primitive (CD34+) (0.93±0.24% versus0.26±0.10%; FIG. 12F) human cells than mice transplanted with STFcultured cells or cells cultured with soluble antibody (FIGS. 12B,12D-12F).

To measure the self-renewal potential of SCID-repopulating cells (SRCs)after culture, bone marrow was collected from the primary mice andtransplanted into sublethally irradiated secondary recipients.Engraftment of secondary recipients with cells cultured in STF medium orin soluble antibody was barely detectable. In contrast, the cellscultured with immobilized antibody showed positive engraftment ofmyeloid, B-lymphoid, and primitive cells after the secondarytransplantation (FIGS. 12G-12L). Similar results were obtained fromanother independent experiment using human CB CD34+ cells for culture(FIGS. 13A-13D).

CD133+ cells cultured with soluble or immobilized anti-LILRB2 mAb werealso transplanted into sublethally irradiated NSG mice (1×10⁴ inputequivalent cells per mouse). In the primary transplantation, engraftmentwith immobilized monoclonal anti-LILRB2 treated cells was detectable butnot significantly different from cells cultured without antibodytreatment or with soluble anti-LILRB2 (FIGS. 14A-14F). In the secondarytransplantation, however, only cells treated with immobilizedanti-LILRB2 showed positive engraftment (FIGS. 14G-14L). Together, thedata indicate a net expansion of HSCs during the initial culture period,and it is thus concluded that immobilized anti-LILRB2 antibodies supportextensive ex vivo expansion of human SRCs.

A limiting dilution assay was performed to quantitate the SRCfrequencies before and after culture. Human CB CD133+ cells culturedwith immobilized anti-LILRB2 antibody-cultured for 10 days had a112-fold increase in total nucleated cells (TNCs) and a 19-fold increasein CD34+ cells relative to input cells (FIGS. 15A-15B). Cultured cellshad greater average chimerism than in peripheral blood (FIG. 15C) andbone marrow (FIG. 15D). As part of the limiting dilution assay, theengraftment by 700-4,000 uncultured CD133+ cells, and the progenies ofthese cells after culture was measured. All mice transplanted with thecultured progenies of 4,000 CD133+ cells engrafted at a level greaterthan 1%. FIG. 15E shows that the frequency of repopulating cells (CRU)from uncultured CD133+ cells is 1 per 4557 cells (95% confidenceinterval for mean: 3222 to 6445, n=24). That is, as calculated fromPoisson statistics, injection of an average of 4,557 cells from this lotof uncultured human CD133+ cells would be sufficient to repopulate 63%(=1−1/e) of transplanted mice. In contrast, the CRU after culture was 1per 932 input equivalent cells (95% confidence interval for mean: 689 to1261, n=24). There was a 4.9-fold increase in the number of SRCs aftercultured in STF medium with immobilized anti-LILRB2 polyclonal antibody.These cultured cells had much greater levels of multi-lineageengraftment than uncultured cells (FIGS. 15F-15I).

It has previously been shown that several Angptls support ex vivoexpansion of HSCs (Zhang et al., Blood. 2008; 111:3415-3423; Zhang etal., Nat Med. 2006; 12:240-245; and Huynh et al., Stem Cells. 2008;26:1628-1635) but the mechanism responsible for this activity wasunknown. Here it has been demonstrated that mammalian-expressed Angptl2exists as HMW species, and ligand multimerization is required foractivation of LILRB2 for downstream signaling. Motifs in the Ig domainsof LILRB2 that are critical for the Angptl2 binding and signalingactivation were also identified. It was shown that the binding ofAngptl2 to LILRB2 is greater than and not completely overlapped with thebinding of another ligand HLA-G. In an attempt to identify agonists ofLILRB2 that are more potent and stable than Angptl to support ex vivoexpansion of human HSCs, it was found that immobilized polyclonalanti-LILRB2 supports consistent ex vivo expansion of human CB HSCs. Thisstudy thus started to uncover the molecular basis for Angptl/LILRB2interaction. It also provides functional evidence that manipulation thebinding between the ligands and LILRB2 on HSCs supports the repopulatingactivity of HSCs, and demonstrated a novel approach for efficientexpansion of HSCs that may find utility in HSC-based cell therapies.

While the bona fide signaling reporter of LILRB2 is not available, achimeric receptor surrogate reporter system was developed that canevaluate the ability of a ligand to bind and activate LILRB2. As shownin the described studies, this reporter cell line can serve as asensitive system to enable comparison of the signaling-inductionabilities of different forms of ligands. This chimeric receptor reportersystem will also be useful to screen additional agonists and antagonistsof ITIM-containing receptors. It is envisioned that the agonists ofITIM-containing receptors may facilitate stem cell-based regenerativemedicine, and the antagonists may serve as inhibitors of cancerdevelopment.

The critical factors that contribute to LILRB2 activation by Angptl2were identified. First, it was determined that mammalian-expressedAngptl2 forms HMW species that appear to be important for its binding tothe receptor. Angptl2 contains both CC and FBN domains. Based on severalpieces of evidence, neither the CC domain nor the FBN domain of Angptl2alone bind to LILRB2 as potently as the full-length Angptl2. Both the CCdomain and full-length LILRB2 exist as HMW species, whereas the FBNdomain does not. Concordantly, a previous study showed that the CCdomain of Angptl4 mediates multimerization. Ge et al., J. Biol Chem.2004; 279:2038-2045. These data suggest that both CC and FBN domainscontribute to the receptor binding and that the CC domain-mediatedmultimerization significantly enhances the binding of the full-lengthAngptl2 to LILRB2. Although Angptls are observed as soluble hormones inserum, they can also be enriched on the plasma membrane in vitro (datanot shown). In addition to the soluble multimerized form, it isspeculated that clustered, cell surface-bound Angptls exist in vivo andactivate LILRB2.

The features of LILRB2 important for Angptl2 binding were furtheridentified. Novel H*G*Y*C motifs in the first and fourth Ig domains ofLILRB2 are essential to Angptl2 binding. The critical necessity of thismotif in Angptl2/LILRB2 binding is supported by the effects of thesite-specific mutations at G94, R95, and Y96 in Ig1 that reduced Angptl2binding by 50%. Similarly, a single mutation in Y394 in Ig4 decreasedAngptl2 binding of full-length LILRB2 by 40%. In addition to Angptls,LILRB2 is known to have other ligands including various MHC class Imolecules. Shiroishi et al., Proc Natl Acad Sci USA. 2003;100:8856-8861. An important question is whether Angptl and MHC class Imolecules bind to LILRB2 in a similar or different manner. As the flowcytometry-based ligand binding assay showed, Angptl2 has a greaterbinding than HLA-G to LILRB2. This result is supported by the describedreporter cell assay. In contrast to Angptl2, a much greater dose of theimmobilized HLA-G cannot induce GFP expression of the LILRB2 reportercells. Therefore, the Angptl2 activation of the LILRB2 chimeric receptorreporter cells is much more potent than HLA-G; however, Angptl2 is notefficiently to compete the HLA-G binding to LILRB2 (data not shown).Whereas HLA-G binds to the first two Ig domains of LILRB2 28, Angptl2binds to both Ig1 and Ig4 of LILRB2. Interestingly, the H*G*Y*C motifthat is critical for Angptl2 binding to LILRB2 is not within either ofthe two MHC binding sites or at the typical interfacial loop region, butwithin the beta-sheet structure, as revealed by a crystallographicstudy. Shiroishi et al., Proc Natl Acad Sci USA. 2006; 103:16412-16417.Based on the described binding and activation results, this motif shouldbe required for the conformational stability of the Ig domains of LILRB2that is needed for binding and activation by Angptl2. Together, theseresults suggest that, 1) Angptl2 binds and activates LILRB2 with agreater ability than MHC class I, and 2) Angptl2 and MHC class Imolecules may not directly compete for binding to the same sites, andthese two types of molecules may be able to act individually or evencooperatively so that the signaling of LILRB2 may be co-regulated.Nevertheless, the binding of each of the two ligands may somewhatrequire common conformational alterations. For example, while themutations of most of the MHC-I binding sites of LILRB2 did not affectAngptl2 binding, it was observed that the mutation G94D, R95E, or Y394Ain LILRB2 that is within the H*G*Y*C motif and decreases Angptl2 bindingalso showed lower HLA-G binding.

Interestingly, while all LILRBs contain a G*Y*C motif, Angptl2 onlybinds LILRB2. This suggests that the H*G*Y*C motif is necessary but notsufficient for maintaining the LILRB2 conformation for Angptl2 bindingand activation.

Based on the described identification of the binding sites of Angptl2 toLILRB2, an improved strategy to use immobilized anti-LILRB2 for ex vivoexpansion of human HSCs was developed. Immobilized antibodies bind tothe same region of LILRB2 as Angptl2. The serum-free culture systemcontaining defined cytokines and immobilized anti-LILRB2 supports a netexpansion of repopulating human CB HSCs, as determined by the serial NSGtransplantation and limiting dilution analysis. The polyclonalanti-LILRB2 antibody demonstrated a greater ability to support ex vivoexpansion of HSCs than monoclonal anti-LILRB2, suggesting thepossibility of multiple ligand binding sites in LILRB2 activation. Inaddition, the internalization signal YXXphi of LILRB2 (Kozik et al.,Traffic. 2010; 11:843-855) suggests that LILRB2 can undergo endocytosis,possibly after ligand binding. Because the immobilized antibodies mayprevent this event and thus prolong the receptor activation, the ex vivoexpansion of HSCs may also be enhanced by the immobilized antibodies.Together, because the anti-LILRB2 polyclonal antibodies are easier to beexpressed and purified and more stable than Angptls, and importantly,bind and activate LILRB2 with a greater ability than Angptl2, thissystem may have greater advantages to use in ex vivo expansion of HSCs.

3.2 Example 2

Previous studies have led to Notch-induced ex vivo expansion ofhematopoietic stem cells (HSC) and their successful use for reducing theprolonged post-transplant neutropenia encountered in patients undergoingCB stem cell transplantation (see FIGS. 16 and 17). More recently arelationship between the number of expanded, partially HLA-matched CD34+cells infused and time to neutrophil recovery has been observed,suggesting a critical need for greater expansion of CB HSPC to reliablyenhance early neutrophil recovery.

To alleviate the risks associated with delayed myeloid recovery, methodsfor ex vivo expansion of CB HSPC that are infused along with one or twonon-manipulated CB units in the setting of myeloablative hematopoieticstem cell transplantation (HSCT) have been developed. These methods arebased on previously described studies of the role for the Notch receptorfamily in regulation of hematopoiesis and on pre-clinical development ofa Notch-mediated expansion system for hematopoietic progenitors usingthe Notch ligand Delta1. Milner et al., Blood. 1994; 83(8): 2057-2062;Varnum-Finney et al., Nat Med. 2000; 6(11): 1278-1281; Varnum-Finney etal., Blood. 2003; 101(5): 1784-1789; Delaney et al., Blood. 2005;106(9): 2693-2699. Clinical translation of this work resulted in a PhaseI CBT trial using ex vivo expanded CB progenitor cells followingmyeloablative conditioning. Results of this trial showed both safety ofthis approach as well as significant decrease in time to neutrophilengraftment. An update of these studies since the initial publication(Delaney et al. Nat Med. 2010; 16(20): 232-237) shows reduction inmedian time to initial ANC≥500/μl of 11 days compared with a concurrentcohort of patients (N=29) with the same treatment regimen (p<0.0001,FIG. 16). Significantly, achievement of ANC>100/μl, an indicator of riskof day +100 mortality (Dahlberg et al. Blood. 2011; 118(21): 3033)occurred at 7 days as compared to 19 days in the conventional double CBTgroup (p=0.0002). Moreover a relationship between number of HSPC infusedand time to engraftment, with 6 out of 7 patients who received greaterthan 8×10⁶ CD34+ cells/kg achieving an ANC≥500/μl within 10 days (FIG.17) was observed, findings unique with respect to cell-therapy basedreduction of neutropenia. This observed dose-relationship is reflectiveof studies in non-manipulated CBT indicating that higher cell doses ofCD34+ cells/kg are required to overcome increased HLA disparity andallow engraftment in partially-HLA matched CBT recipients. Laughlin etal. N Engl J Med. 2001; 344(24): 1815-1822; Wagner et al. Blood. 2002;100(5): 1611-1618.

Aspects of the current disclosure are based on the hypothesis thatinhibition of differentiation by induction of endogenous Notch signalingin combination with factor(s) able to enhance stem self-renewal and/orsurvival will lead to generation of greater numbers of rapidlyrepopulating CD34+ HSPC. One such factor is angiopoietin agonistsincluding, for example, Angptl5. Angptl5 a member of theAngiopoietin-like family of proteins previously shown to enhance thegeneration of hematopoietic repopulating cells in preclinical studies.

For example, in vitro culture with Angptls 2, 3, 5, and 7 and cytokinessignificantly increases repopulating activities of murine long-term HSC.Zhang et al., Nat Med. 2006; 12(2): 240-245. Culture of CB HSPC withANGPTL5 and growth factors (SCF, TPO, FGF-1, heparin, and IGFBP2) led tosignificantly improved in vivo reconstitution in NOD/SCID mice at 2months post-transplant (39.5% engraftment v. 0.2-2% in the non-culturedgroup, FIG. 18) as well as enhanced secondary transplantation. Zhang etal. Blood. 2008; 111(7): 3415-3423. Moreover, whereas Notch signalinginhibits differentiation allowing HSPC self-renewal, Angptl proteinsenhance in vivo HSPC repopulation in the absence of in vitro evidencedemonstrating altered differentiation or HSC expansion. Aspects of thecurrent disclosure are accordingly based on the hypothesis that becausethese physiologic ligands function by two distinct mechanisms, thecombination may result in generation of more effective repopulatingcells. This hypothesis was tested in Example 2 to further examinewhether combined effects of Delta1- and Angptls, such as Angptl5,enhance HSC expansion, allowing improvement over current cellulartherapies relevant for clinical applications.

Based in part on the described data and hypotheses, the Notch agonistDelta 1 in combination with Angptl5 was selected for further study.However, culture conditions optimized for Delta^(Ext-IgG) and Angptl5are quite distinct. Optimal Delta^(Ext-IgG)-induced expansion was foundfollowing 16-17 days culture in StemSpan serum-free media in thepresence of SCF, Flt3-ligand (Flt3L), TPO, IL-6 and IL-3, withimmobilized Delta^(Ext-IgG) and retronectin. By contrast,Angptl5-induced expansion was optimal following 10 days culture inStemSpan media, but with low-doses of the cytokines SCF, TPO as well asFGF1, heparin, and IGFBP2. Accordingly, success of the combination couldnot be predicted.

3.2.1. Example 2—Methods

Cell Isolation. Human CB for research was obtained from normaldeliveries under Swedish Medical Center Institutional Review Board(Seattle) approval after consent was obtained. Samples were incubated inammonium chloride red blood cell lysis buffer, washed, and suspended inphosphate-buffered saline (PBS) with 2% human type AB serum. Cells wereincubated with CD34+ immunomagnetic beads (Miltenyi Biotec) and purifiedusing a Miltenyi AutoMACS and then frozen. CD34+ cells were then thawedfor individual experiments.

Generation and immobilization of Delta^(Ext-IgG) protein and LILRB2antibody (anti-CD85d). Generation of the construct encoding theextracellular domain of Delta1 fused to the fc domain of human IgG1 andpurification of Delta^(Ext-IgG) protein from culture medium of NSO cellselectroporated with the construct have been previously described(Varnum-Finney, J Cell Sci. 2000; 113(pt23): 4313-4318). Human CD85dbiotinylated antibody (polyclonal goat IgG) or normal goat IgGbiotinylated control were obtained from R&D Systems. Wells of non-tissueculture-treated culture plates or non-tissue culture-treated flasks wereincubated with Delta^(Ext-IgG) (0.5 or 2.5 μg/ml), CD85d biotinylatedantibody (range 0.08 to 25 μg/ml), or IgG control diluted in PBStogether with 5 μg/ml retronectin, incubated overnight at 4° C., andwashed generously with PBS.

Cell Cultures. Cells were cultured in serum-free medium (Stemspan SerumFree Expansion Medium; StemCell Technologies) with 50 ng/ml human stemcell factor (SCF), human Flt3-ligand, human interleukin 6 (IL-6),thrombopoietin (TPO), 10 ng/ml human interleukin 3 (IL-3), FGF1 20ng/ml, and heparin 10 μg/ml. Cultures for transplant were initiated in25 cm² non-tissue cultured treated flasks with between 1 and 1.2×10⁵starting CD34+ cells/flask. Cells were expanded to 75 and 125 cm²non-tissue cultured treated flasks when cell density reachedapproximately 1-1.5×10⁶ cells/ml. Fresh medium with cytokines was addedevery 3-4 days.

Transplantation of human hematopoietic cells into NOD/SCID gamma nullmice. Sublethally irradiated (275 rad) NOD-SCID IL-2Rγ-null mice (NSG)approved for use by the Fred Hutchinson Cancer Research CenterInstitutional Animal Care and Use Committee were used for transplant.Single mice (5-8 mice/group) were infused with the progeny generatedfrom 1×10⁵ started CD34+ cells. Repopulating ability (percent humanCD45+ or CD45+33+, CD 45+19+, CD45+34+ in marrow) was assessed at 2 and8 weeks after transplantation using bone marrow aspirated from the femurof anesthetized recipient mice.

Statistical Analysis. Unpaired, two-tailed t-tests were used to makecomparisons between groups

3.2.2. Example 2—Results

The combined effects of Delta1 and Angptl5 enhanced generation ofCB-derived CD34+ cells that rapidly repopulate the marrow of immunedeficient mice with hematopoietic precursors and differentiating myeloidcells. Particularly, in studies combining Delta1 and Angptl5,significantly enhanced rapid (2 week) repopulation of NSG mice by CD34+precursors (p=0.04) and a clear trend towards enhanced repopulation byimmature, CD33+ myeloid precursors (p=0.08 two-tailed t-test, p=0.04Mann-Whitney test for possible non-Gaussian distribution) was foundcompared to using Delta1 or Angptl5 alone (FIG. 19). Importantly, thisoccurred despite overall similar CD34-fold expansion with CB HSPCcultured on Delta1 alone (FIG. 20). Additionally, there was nodifference in generation of CD34+CD90^(lo) or CD49f cells suggestinggeneration of greater numbers of rare, rapidly repopulating cells, orcells with altered cell-cycle, apoptotic, or transcriptional orepigenetic properties. Of note, this combination maintainedmulti-lineage engraftment 8 weeks after transplant suggestingmaintenance or expansion of longer term repopulating cells.

FIG. 21 shows significantly enhanced early marrow repopulation whenDelta is combined with Angptl5 and cultured in conditions optimized forDelta-mediated expansion.

FIG. 22 shows longer-term repopulation is significantly enhanced whenDelta is combined with Angptl5 and cultured in conditions optimized forDelta-mediated expansion; repopulation is multi-lineage showingsignificantly enhanced myeloid and lymphoid lineages. Cells did not havesignificant secondary engraftment when cultured in conditions previouslyoptimized for Delta-mediated expansion.

FIGS. 23A and 23B show that culture with Delta and Angptl5 with lowercytokine concentrations results in secondary engraftment previously notseen in Delta expanded cells suggesting maintenance/expansion of alonger-term repopulating cell when Delta is added to Angptl5 in theseconditions.

FIG. 24. Culture with Delta and an antibody to the Angptl5 receptor(LILRB2 or CD85) trends towards enhanced early myeloid engraftment ascompared to Delta alone. This trend is present at the highest dose ofCD85 used in this experiment.

FIG. 25. When engraftment was assessed at a longer-term time point (16wks after transplant), engraftment of cells cultured with Delta and CD85had greater engraftment than Delta alone. These cells are able torepopulate both lymphoid and myeloid lineages.

Accordingly, Example 2 demonstrates the development of cultureconditions for expanding HSC using Delta1 and Angptl5 with cytokinecompositions and time in culture. Significantly enhanced early marrowrepopulation in immune-deficient mice from CB hematopoieticstem/progenitor cells (HSPC) cultured with Delta1 and Angptl5 ascompared to Delta1 alone is demonstrated. Further, data demonstratingimproved longer-term repopulation of CB HSPC following culture withDelta1 and Angptl5 as compared to either approach alone is provided.

3.3 Example 3

The focus of this Example is on optimizing generation of CBhematopoietic stem/progenitor cells (HSPC) by investigating not onlyculture conditions but also presentation of Delta1 and anti-LILRB2antibody.

Use of an immobilized antibody against LILRB2 (CD85d), anAngiopoietin-like (ANGPTL) protein receptor, was investigated.Interaction of LILRB2 and ANGPTL protein allows for the ex vivoexpansion of CB HSPC, including those with in vivo repopulating ability.In initial studies, combination of immobilized Delta1 with anintermediate concentration of anti-LILRB2 antibody led to the increasedgeneration of CD34+ and CD34+CD90^(lo) cells (CD90^(lo) cells were thosecells expressing low amounts of CD90, as determined by a gated cut-offamount) compared to culture with Delta1 or anti-LILRB2 antibody alone(data not shown). Transplantation experiments into NSG mice to optimizeculture conditions for maximal generation of rapid myeloid repopulatingcells demonstrated improved generation of cells using previouslyestablished cytokine culture conditions and immobilized anti-LILRB2antibody at concentration of 1.25 μg/ml (data not shown). Upontransplant into NSG mice, the combined activation of Notch and LILRB2receptors enhanced the generation of rapidly repopulating myeloidprecursors compared to Delta1 alone (29.3% vs. 18.6% total human CD33+cells in NSG mice at 2 weeks post-transplant, p=0.05) (FIG. 26). Culturein these conditions may also enhance generation of longer-termrepopulating cells as shown by greater progenitor cell engraftment withthe combination (2.9% vs. 1.7% total human CD34+ cells in NSG mice at 10weeks post-transplant) (FIG. 27).

Enhanced Hes1 (a Notch target gene) expression was observed in cellscultured with the combination of Delta1 and anti-LILRB2 antibody overDelta1 alone, with all reagents immobilized on the plastic of theculture dish (FIG. 28).

Studies investigating alternate methods of agonist presentation wereperformed, specifically comparing the effectiveness of agonistimmobilized on magnetic protein A-microbeads with immobilization to theculture flask. Data suggest that presentation of Delta1 and anti-LILRB2antibodies induced greater Hes1 expression when the two agonists werepresented by alternate means, e.g. Delta1 immobilized on plastic andanti-LILRB2 antibody immobilized on beads (FIG. 29).

Amplification in the production of CD7+ cells accompanying the increasedHes1 expression has not been observed, suggesting that the expression ofT cell genes are unaffected. Without being bound by any particulartheory, these data suggest that the activation of the ANGPTL5/LILRB2pathway is not amplifying the effects of Notch signaling, but acts inparallel with Notch, perhaps at the Hes1 gene level, to inhibit myeloiddifferentiation and enhance HSPC self-renewal.

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of orconsist of its particular stated element, step, ingredient or component.Thus, the terms “include” or “including” should be interpreted torecite: “comprise, consist of, or consist essentially of.” As usedherein, the transition term “comprise” or “comprises” means includes,but is not limited to, and allows for the inclusion of unspecifiedelements, steps, ingredients, or components, even in major amounts. Thetransitional phrase “consisting of” excludes any element, step,ingredient or component not specified. The transition phrase “consistingessentially of” limits the scope of the embodiment to the specifiedelements, steps, ingredients or components and to those that do notmaterially affect the embodiment. As used herein, a material effectwould cause a statistically significant reduction in precursor cellexpansion by the methods disclosed herein as measured by an assaydescribed in relation to effective amounts.

Unless otherwise indicated, all numbers used in the specification andclaims are to be understood as being modified in all instances by theterm “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. When furtherclarity is required, the term “about” has the meaning reasonablyascribed to it by a person skilled in the art when used in conjunctionwith a stated numerical value or range, i.e. denoting somewhat more orsomewhat less than the stated value or range, to within a range of ±20%of the stated value; ±19% of the stated value; ±18% of the stated value;±17% of the stated value; ±16% of the stated value; ±15% of the statedvalue; ±14% of the stated value; ±13% of the stated value; ±12% of thestated value; ±11% of the stated value; ±10% of the stated value; ±9% ofthe stated value; ±8% of the stated value; ±7% of the stated value; ±6%of the stated value; ±5% of the stated value; ±4% of the stated value;±3% of the stated value; ±2% of the stated value; or ±1% of the statedvalue.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to publications, patentsand/or patent applications (collectively “references”) throughout thisspecification. Each of the cited references is individually incorporatedherein by reference for their particular cited teachings.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

Definitions and explanations used in the present disclosure are meantand intended to be controlling in any future construction unless clearlyand unambiguously modified in the examples or when application of themeaning renders any construction meaningless or essentially meaningless.In cases where the construction of the term would render it meaninglessor essentially meaningless, the definition should be taken fromWebster's Dictionary, 3^(rd) Edition or a dictionary known to those ofordinary skill in the art, such as the Oxford Dictionary of Biochemistryand Molecular Biology (Ed. Anthony Smith, Oxford University Press,Oxford, 2004).

Various references such as patents, patent applications, andpublications are cited herein, the disclosures of which are herebyincorporated by reference herein in their entireties.

1. A method of expanding hematopoietic stem cells and/or hematopoieticprogenitor cells ex vivo, comprising culturing the cells with: (i) aNotch agonist; and (ii) a leukocyte immunoglobulin-like receptor B2(LILRB2) agonist immobilized on a first solid phase, wherein theimmobilized LILRB2 agonist is an antibody to the LILRB2 receptor, or anantigen-binding fragment of said antibody.
 2. The method of claim 1,wherein the culturing comprises adding fresh media every 3 or 4 days fora period of 10 to 20 days.
 3. The method of claim 1, wherein theculturing is within a serum-free culture media.
 4. The method of claim1, wherein the culture media comprises 10-100 ng/mL stem cell factor(SCF), 5-100 ng/mL thrombopoietin (TPO), 10-100 ng/mL Flt3 ligand(Flt3-L), and 1-100 μg/mL of retronectin.
 5. The method of claim 1,wherein the hematopoietic stem cells and/or hematopoietic progenitorcells are human cells obtained from bone marrow, umbilical cord blood,placental blood, or Wharton's jelly.
 6. The method of claim 1, whereinthe cells are hematopoietic stem cells or hematopoietic progenitorcells.
 7. The method of claim 1, wherein the cells are hematopoieticstem cells and hematopoietic progenitor cells.
 8. The method of claim 1,wherein the LILRB2 agonist is an Fv, Fab, Fab′, F(ab′)₂, or single chainFv fragment (scFv).
 9. The method of claim 1, wherein the LILRB2 agonistis immobilized on the first solid phase at a concentration of 0.08 to 25μg/mL.
 10. The method of claim 1, wherein the Notch agonist isimmobilized on a second solid phase at a concentration of 0.025 to 5μg/mL.
 11. The method of claim 1, wherein the Notch agonist isDelta^(ext-IgG).
 12. The method of claim 1, wherein the Notch agonist isan antibody that specifically binds to Notch-1 or an antibody thatspecifically binds to Notch-2.
 13. A method of expanding hematopoieticstem cells and/or hematopoietic progenitor cells ex vivo, comprisingculturing the cells with: (i) a Notch agonist; and (ii) a leukocyteimmunoglobulin-like receptor B2 (LILRB2) agonist immobilized on a firstsolid phase, wherein the immobilized LILRB2 agonist is Angiopoietin-like5 (Angptl 5).
 14. The method of claim 13, wherein the culturing is in aserum-free media comprising 1-100 ng/mL stem cell factor (SCF), 1-100ng/mL thrombopoietin (TPO), 1-100 ng/mL FLt-3 ligand (Flt3-L), 1-100ng/mL interleukin-6 (IL-6), 1-100 ng/mL interleukin-3 (IL-3), 1-100ng/mL FGF1, and 1-100 μg/mL heparin.
 15. The method of claim 13, whereinAngptl 5 is immobilized on the first solid phase at a concentration of0.08 to 25 μg/mL.
 16. A chimeric reporter system for screening agonistsand antagonists of leukocyte immunoglobulin-like receptor B2 (LILRB2)comprising: cells expressing: a fusion protein comprising at least oneextracellular domain of LILRB2 and transmembrane and cytoplasmic domainsof paired immunoglobulin-like receptor β (PILRβ); and an adapter proteinthat associates with the cytoplasmic domain of the fusion protein andactivates a transcription factor, and a reporter gene within the cellsresponsive to the transcription factor.
 17. The system of claim 16,wherein the fusion protein is encoded by a gene on a retroviral vector.18. The system of claim 16, wherein the cells are mouse T cell hybridomacells.
 19. The system of claim 16, wherein the transcription factor isnuclear factor of activated T cells (NFAT).
 20. The system of claim 16,wherein the adapter protein is DAP12.